A rapid and efficient platelet purification protocol for platelet gene

expression studies

Stefan Amisten

Address: Oxford Centre for Diabetes, Endocrinology and Metabolism, Oxford University, The
Churchill Hospital, Headington, Oxford, OX3 7LJ, U.K.



Email: stefan.amisten@gmail.com




Abstract
Isolation of pure platelet samples from whole blood is crucial for the study of platelet gene
expression. The main obstacles to overcome in order to successfully isolate platelets from whole
blood include: 1) platelet activation; 2) leukocyte and red blood cell contamination and 3) time-
dependent platelet mRNA degradation. This chapter describes a rapid and highly efficient method for
isolating human circulating platelets from small volumes of whole blood based on efficient inhibition
of platelet activation and leukocyte removal by filtration followed by magnetic bead-depletion of
residual contaminating leukocytes and red blood cells. Also described are methods for RNA
extraction, cDNA synthesis and platelet gene expression studies using both quantitative real-time
PCR and microarray.

Key Words: platelet purification, platelet activation, leukocyte-depletion, gene expression, real-time
PCR, qPCR, microarray, RNA extraction.

1. Introduction
Microarray and real-time PCR have both been used to study the platelet transcriptome(1-5).
However, successful platelet gene expression profiling is often hampered by the contamination of
platelet preparations by other cell types such as leukocytes.

Platelets are anuclear and, as a consequence, all non-mitochondrial platelet mRNA is derived from
their precursor cell, the megakaryocyte. As a result, the amount of mRNA a platelet contains is
dependent on the amount of mRNA inherited when it budded off from the megakaryocyte as well as
the rate of platelet RNA degradation. Therefore, when studying platelet gene expression, it is
advantageous to use a fast platelet purification method in order to minimize further degradation of
mRNAs during the purification process. Also, it is important to minimize the contamination of the
platelet preparations by other cell types such as red blood cells and leukocytes. This is particularly
important for leukocytes, which have been reported to contain as much as 10,000 times more RNA
than platelets(6).

This chapter describes a fast, flexible and low cost method for the generation of a highly purified
population of human platelets from less than 100 mL of whole blood(1) (see Note 1). The protocol
combines efficient inhibition of platelet activation with leukocyte removal using filters developed for
the preparation of platelets for clinical use (7) and antibody-mediated magnetic depletion of residual
leukocytes and erythrocytes. The purification can be performed in less than two hours and without
the need for expensive or bulky specialized equipment. The chapter also describes the use of real-
time PCR to assess the expression of two platelet P2Y receptors (P2RY1 and P2RY12) and the level of
residual contaminating leukocyte-specific messages (CD45).

2. Materials

2.1 Platelet isolation
1. Dynabeads Pan Mouse IgG (Invitrogen)
2. Mouse anti-human CD235a (red blood cell surface marker) (BD Inc, NJ, USA)
3. Mouse anti-human CD45 (leukocyte surface marker) (BD Inc, NJ, USA)
4. Dynabead-compatible magnet (DynaMag-2 or DynaMag-15) (Invitrogen) PBS, pH 7.4
(Invitrogen) (see Note 2)
5. Dynabead wash buffer: PBS with 0.1% (w/v) BSA, pH 7.4
6. Sterile anticoagulant: citrate-dextrose solution (ACD) (Sigma Aldrich)
7. Prostaglandin E1 (PGE1) (Sigma Aldrich): 1 mM stock in ethanol
8. Acetylsalicylic acid (Sigma Aldrich): 30 mM stock in ethanol
9. EDTA, 0.5M (Sigma Aldrich)
10. 50 and 15 mL centrifuge tubes and 1.5 and 2 mL Eppendorf tubes (see Note 2)
11. Intravenous venflon cannula 18 gauge with port and flexible tubing (Becton Dickinson
Infusion Therapy AB, Sweden)
12. Rotating and tilting test tube carousel
13. AutoStop BC high efficiency filter for leukocyte removal (Pall Inc, Cat no ATSBC1E, see
http://www.pall.com/medical_48187.asp)
14. TRIzol (Invitrogen)

2.2 RNA purification and cDNA generation
1. RNase free filter tips: 10 L, 100 or 200 L and 1000 L
2. RNase free tubes 1.5 mL and 0.6 mL (Axygen MCT-060-A and MCT-150-C)
3. Isopropanol, molecular biology grade (SigmaAldrich)
4. Ethanol, molecular biology grade (SigmaAldrich)
5. Water, molecular biology grade (ie. RNase-free) (SigmaAldrich)
6. Chloroform, molecular biology grade (SigmaAldrich)
7. UltraPure Glycogen (Invitrogen, Cat. No. 10814-010)
8. PCR tubes, RNase free
9. TaqMan reverse transcription kit (Applied Biosystems)
10. RNeasy MinElute Cleanup Kit (Qiagen)
11. Centrifuge with cooling for spinning at 12000 x g
12. NanoDrop (Thermo Scientific) or equivalent spectrophotometer for assaying nucleotide
content in small sample volumes
13. Optional - 2100 Bioanalyzer (Agilent Technologies)

2.3 Quantitative real-time PCR
1. QuantiFast SYBR Green PCR Kit (Qiagen)
2. QuantiTect Primer Assays (Qiagen):
a. P2RY1 (QT00199983) platelet ADP receptor used to demonstrate the qPCR protocol
b. P2RY12 (QT01770111) platelet ADP receptor used to demonstrate the qPCR protocol
c. ACTB (QT01680476) housekeeping gene
d. P2RY11 (QT01150282) GPCR expressed on leukocytes but not on platelets
e. CD45 (PTPRC) (QT01869931) abundant leukocyte protein, not present on platelets
3. PCR tubes
4. Filter tips, PCR grade
5. Equipment for agarose gel electrophoresis of 50 200 bp qPCR products

3. Methods
The platelet purification method has four main phases: blood collection, generation of platelet rich
plasma (PRP), PRP filtration and magnetic bead-mediated leukocyte and erythrocyte depletion.

Appropriate ethical permission to take human blood should be obtained. To minimize platelet
activation during blood collection, a 1.2 mm diameter (18 gauge) intravenous cannula is inserted into
a suitable vein, and the blood is collected through the attached tubing via gravity flow into the
platelet inhibition cocktail. In the authors experience, this method is superior to collecting blood
using vacutainers, as it reduces mechanical platelet activation caused by the vacuum. Once purified,
the platelet samples are dissolved in TRIzol and frozen until RNA extraction.

3.1. Preparation of antibody-conjugated magnetic beads
1. Mix 1 mL Dynabead wash buffer and 250 l Dynabead slurry.
2. Place the tube in the Dynamag magnet for 1 min, remove the liquid and repeat for a total of
two washes.
3. Remove the washed beads from the magnet and resuspend the washed Dynabeads up to the
original bead volume (i.e. 250 L) using Dynabead wash buffer
4. Add 15 l of anti-CD235a antibody and 15 l anti-CD45 antibody to the beads and mix.
5. Incubate the bead mix for at least 30 min with gentle tilting and rotation.
6. Place the tube in the magnet for 1 min and remove the supernatant. Resuspend the beads up
to the original bead volume (i.e. 250 L) using Dynabead wash buffer and store the antibody-
conjugated magnetic beads at 4
o
C.

3.2. Preparation of platelet inhibition cocktail
The volumes stated below apply to 100 mL whole blood collected into twelve 15 mL tubes.
1. Mix 18 mL ACD, 12 l of 1 mM PGE
1
, 120 l of 30 mM Acetylsalicylic acid and 480 l of 0.5M
EDTA
2. Add 1.5 mL of the cocktail into each of the twelve 15 mL tubes

3.3. Collection of blood and purification of platelets
1. Weigh an empty 15 mL tube with cap and note down the tube weight.
2. Implant the intravenous cannula (1.2 mm diameter) and attach the flexible tubing. Collect 8.5
mL of blood by self propagated flow through the flexible tubing into each tube containing 1.5
mL platelet inhibition cocktail. When the total volume reaches 10 mL, replace the cap and
invert the tube gently to enable thorough mixing of the blood with the platelet inhibition
cocktail. To fill 12 tubes you will need 102 mL of whole blood (see Notes 1, 3-4).
3. Place tubes in a centrifuge and spin at room temperature (200 x g, 20 min)
4. Carefully transfer the top 85% of the upper layer, the platelet rich plasma (PRP), to new 15
mL tubes.
5. Spin at room temperature (200 x g, 10 min) to further remove leukocytes and red blood cells.
6. Carefully transfer the top 85% of the PRP to a 50 mL tube.
7. With a sterile pair of scissors or a surgical blade, cut off excess tubing from the Pall Leukocyte
removal filter pack and mount it on a rack.
8. Place the drainage tubing in a 50 mL collection tube and manually inject the PRP into the
filter at a flow rate of 15 mL per minute. Collect the filtrate in a 50 mL tube.
9. Add all of the antibody-conjugated magnetic beads to the tube containing the filtered PRP
and place the tube in a tilting and rotating carousel at room temperature for 45 minutes.
10. Transfer the PRP/bead mix into 2 mL tubes (see Note 2) and leave the tubes in the
DynaMag-2 for 2 minutes. The DynaMag-2 can accommodate 16 tubes at a time, so steps
10-13 will have to be repeated if more than 32 mL of PRP is used.
11. With the tubes still held in the magnetic rack, transfer the supernatants into new magnet
compatible tubes without disturbing the magnetic beads that are held by the magnetic field.
12. Put the tubes containing the depleted PRP in the magnet.
13. After 2 min, without disturbing the beads captured by the magnetic field, transfer the
supernatant into new 15 mL tubes and harvest the platelets by centrifugation at 800 x g, 10
minutes at room temperature
14. Discard the supernatants. Weigh the 15 mL tubes containing the platelet pellets and
calculate the weight of each platelet pellet by subtracting the empty tube weight from step 1
above. Dissolve the platelet pellets using 1 mL TRIzol per 100 mg platelets (use a minimum of
1 mL TRIzol for platelet samples weighing less than 100 mg) and leave for 5 minutes at room
temperature (see Note 5).
15. Transfer the dissolved platelets into 1.5 mL tubes (1 mL per tube) and store the samples at -
80 C until RNA purification. RNA samples in TRIzol are stable for up to 1 month at -80 C or
below.

3.4. Extraction of platelet total RNA
The following protocol for isolation of platelet total RNA is based on a modified version of the TRIzol
method(1). An advantage of the TRIzol method over column-based RNA purification methods is that
besides high quality RNA, platelet protein may also be extracted from the same platelet TRIzol
sample (see Notes 5-9). The RNA isolated using TRIzol is then further purified and concentrated using
an RNeasy MinElute Cleanup Kit (Qiagen). The RNeasy MinElute Cleanup Kit columns remove
contaminating residual salts and phenol as well as RNAs shorter than 200 base pairs, such as 5.8S
rRNA, 5S rRNA, and tRNAs, which together comprise 1520% of the total RNA, resulting in an
enrichment of mRNAs(8).

1. Cool isopropanol to -20 C.
2. Cool centrifuge to 4 C.
3. Place platelet samples dissolved in TRIzol in a fume hood, defrost and leave for 5 min at
room temperature.
4. Add 200 l chloroform to each tube containing 1 mL TRIzol (see Notes 5 and 10).
5. Manually shake the tubes 15-30 times and leave for 2-3 min at room temperature
6. Spin at 12000 x g for 15 min at 4 C
7. Add 10 g ultra-pure glycogen (0.5 l of 20 g/L stock solution) to new RNase free tubes
(see Note 11)
8. Transfer the aqueous phase of each tube containing TRIzol/chloroform mix to tubes
containing glycogen using a 1000 L filter tip. Leave about 3-4 mm of the aqueous phase
above the interphase (figure 1) to minimize carryover of contaminating DNA.
9. Add 500 l of cooled (-20 C) isopropanol to each tube of aqueous phase/glycogen mix
10. Invert to mix and leave over night at -20 C or at -80C for 1 hour
11. Spin the samples at 12000 x g for 15 min at 4 C
12. Remove all the liquid and add 1 mL of 75% ethanol (freshly made up with molecular biology
grade water). RNA pellets from the same donor that were split into separate 1 mL aliquots in
TRIzol (steps 14 and 15, section 3.3) may be combined by pooling the RNA pellets into one
tube in a total volume of 1 mL 75% ethanol. RNA samples in 75% ethanol can be stored at -
20 C for at least one year.
13. Spin at 7500 x g for 10 min at 4 C
14. Remove all the liquid. It is important to remove ALL the liquid, otherwise it will take a very
long time to dry the RNA pellet.
15. Dry the pellets by placing the tubes in a heating block at 35-37 C for 1-3 min with the lids
open.
16. When the RNA pellet appears dry, dissolve the RNA in 101 L water, vortex and keep on ice.
17. Transfer 1 L RNA into an RNase-free 1.5 mL tube containing 3 L RNAse free water (makes 4
L of 1 in 4 diluted RNA). Freeze the remaining 100 L RNA at -80C until RNeasy MinElute
Cleanup column purification.
18. Use 1 L of the 1 in 4 diluted RNA to measure the total RNA concentration on a NanoDrop or
equivalent spectrophotometer.
19. Calculate the total amount of RNA present in each sample and purify each RNA sample using
the RNeasy MinElute Cleanup kit according to the manufacturers instructions. Do not load
more than 45 g RNA dissolved in 100 L RNase free water on each spin column(8).
20. The total elution volume from the RNeasy MinElute column is 12 L. Transfer 0.5 L of the
RNeasy MinElute kit purified RNA into a new RNase free tube containing 1.5 L molecular
grade water (makes 2 L of 1 in 4 diluted purified RNA). Store the remaining 11.5 L of RNA
at -20
o
C to -80
o
C until cDNA synthesis.
21. Use 1 L of the 1 in 4 diluted RNA to measure the RNA concentration on a NanoDrop or
equivalent. Calculate the concentration of the undiluted purified RNA sample. The purified
RNA can be used for both cDNA synthesis and microarray hybridization. However, it is
recommended that the RNA integrity of all samples intended for microarray hybridizations
are verified using a 2100 Bioanalyzer (Agilent Technologies).

3.5. Generation of platelet cDNA
Reverse transcription of RNA into cDNA can be achieved using a variety of kits. In our hands, the
TaqMan reverse transcription kit (Applied Biosystems) has worked very well, and it will be used as an
example below. The TaqMan reverse transcription kit is designed to transcribe 60-2000 ng of total
RNA into cDNA. If larger amounts of RNA are needed, it is recommended that several cDNA reactions
not exceeding 2000 ng are performed, followed by pooling of all the cDNA reactions into one.

1. Program the thermal cycler for reverse transcription PCR (table 1).
2. Prepare a reverse transcription master mix (table 2) and distribute to RNase and DNase free
PCR tubes (see Note 12). Add 60-2000 ng RNA to each tube, vortex and spin down. Always
keep all samples and reagents on ice. The total reaction volume can be scaled up or down
according to the amount of cDNA needed.
3. Place PCR tubes containing reverse transcription mix and RNA in the thermal cycler. Ensure
that the PCR tube caps are tightly closed.
4. Once the thermal cycling is completed, the cDNA can be stored short-term at 4 C or long
term at -20 C

3.6 Relative quantitative real-time PCR (qPCR) of platelet genes

To illustrate the use of quantitative real-time PCR (qPCR) the following protocol describes the
quantification of the platelet ADP receptor genes P2RY1 and P2RY12 relative to the house keeping
gene beta-actin (ACTB). The approach uses a Rotor-Gene 6000 real-time PCR cycler, the QuantiFast
SYBR Green PCR Kit and QuantiTect primer assays (all from Qiagen) (see Note 13-14). To evaluate the
degree of residual leukocyte contamination, two leukocyte specific genes may also be quantified:
PTPRC, which encodes the leukocyte surface protein CD45, and P2RY11, which encodes a G-protein
coupled receptor that is expressed on leukocytes but not on platelets. The degree of leukocyte
depletion in the purified platelet samples can easily be measured by qPCR quantification of CD45 in
cDNA from whole blood or PRP (from step 6 in section 3.3) compared to purified platelets from step
13 in section 3.3).

1. Program the Rotor-Gene 6000 thermal cycler (table 3 and Note 15).
2. Prepare a master mix for pilot qPCR quantification (table 4 and Note 16). Keep all samples on
ice and avoid exposing your mastermix to light, as the SYBR Green in the mastermix is light
sensitive. Increased qPCR sensitivity and reduced reaction cost can be achieved by reducing
the total reaction volume from 25 L (i.e. the volume recommended by Qiagen) to 10 or 5 L
without reducing the total amount of cDNA template added to each qPCR reaction (see Note
16). For the pilot qPCR it is recommended to pool cDNA from each biological replicate and to
dilute the pooled cDNA at least 1 in 4, as this approach will use up less cDNA from each
biological replicate.

Example: Platelet cDNA has been generated from 3 different platelet donors. For the initial
pilot qPCR of the genes P2RY1, P2RY12 and ACTB, 1 L of 1 in 4 diluted pooled cDNA will be
used for each 10 L reaction. It is very hard to predict the optimal cDNA dilution for each
gene, since the expression of two different genes can vary by several orders of magnitude.
Therefore, by running a pilot qPCR using a standard dilution it is possible to determine the
optimal cDNA dilution factor required for the quantification of each gene of interest. In order
to include cDNA from all the three platelet samples, 0.5 L cDNA is pooled from each platelet
sample, giving a total volume of 1.5 L pooled platelet cDNA. For a 1 in 4 dilution of the
cDNA, 4.5 l water is added, resulting in 6 L of 1 in 4 diluted pooled platelet cDNA. 3.5 L of
this cDNA is used for the pilot qPCR (see table 4), and the remaining is frozen for future use.

3. Close the lids tightly, place the samples in the thermal cycler and run the program set up in
step 1.
4. Perform a melt curve analysis for each primer assay to verify that only one amplification
product has been produced in each qPCR sample. Please see the product documentation
included with the QuantiFast SYBR Green PCR Kit and your qPCR thermal cycler for specific
instructions on how to perform a melt curve analysis. If a gene of interest is not expressed in
your sample, or if it is present at only very low levels, the QuantiTect primer assay may
generate a non-specific qPCR product (figure 2b). These non-specific amplifications will be
detected by the melt curve analysis and by agarose gel electrophoresis of the qPCR samples.
Due to the small size of the qPCR products (50-200 bp), a 2% agarose gel is recommended for
clear resolution of the individual bands. Only primer assays that generate a single melt curve
peak (Fig. 2a) and a single qPCR product corresponding to the size given in the QuantiTect
primer assay product literature should be used.
5. Calculate the Ct (Cycle threshold) values for all the samples in the qPCR run. Please see the
product documentation included with the QuantiFast SYBR Green PCR Kit and your qPCR
thermal cycler for specific instructions on how to calculate the Ct values. In our hands, when
using the Rotor-Gene 6000 and the RotorGene version 6.0 software, threshold values
between 0.02 and 0.05 often give satisfactory Ct values.
6. Determine the optimal dilution of your cDNA samples for the quantification of your genes of
interest. By diluting the cDNA, it is possible to save platelet cDNA without compromising the
quality of the qPCR quantifications (see Note 17).

Example: The pilot quantification using pooled cDNA diluted 1 in 4 (see steps 2-5 above)
returned the following Ct values: P2RY1: 25.91; P2RY12: 23.26; ACTB: 18.79. Since all Ct
values are between 17 and 30 cycles, no further dilution of the cDNA is necessary, and a 1 in 4
dilution of the cDNA is sufficient for the quantification of all three genes. However, if there is
a need to conserve cDNA, the cDNA used to quantify ACTB can be diluted up to 16 times more
and P2RY12 twice more without compromising the qPCR quality.

7. An ideal primer pair has an amplification efficiency (E) of 2 (i.e. each PCR cycle results in a
doubling of the number of template DNA molecules). However, sometimes the E value
deviates slightly from the theoretical value. In order to determine the actual amplification
efficiency for each primer assay, each gene needs to be quantified using serially diluted
cDNA. The Ct values from the dilution series is then used to calculate the amplification
efficiency for each gene:

E = 10
(-1/k)


where k is the slope of the linear function of the Ct values plotted against the log
transformed cDNA dilution factors. Most functional primer assays will generate primer
efficiencies (E) of 1.9-2.1 (figure 3a), whereas non-functional primer assays will not generate
straight Ct vs. log dilution factor curves (figure 3b) or primer efficiencies much greater or
smaller than 2.

To determine the E value for a primer assay, prepare serially diluted cDNA (see Table 7) and
quantify each gene in triplicate as described in steps 2-6 above. For each gene, prepare a
master mix for 17 x 10 L qPCR reactions (see table 5).

Example: The primer efficiency (E) for the primer assays P2RY1, P2RY12 and ACTB needs to be
determined experimentally using serially diluted platelet cDNA. The required volumes for the
evaluation of three primer assays in triplicate can be calculated as shown in table 6 and table
7.

The average Ct values for each dilution step are plotted in a graph against the log values of
the dilution factors and the equation for the linear trendline is calculated (table 8 and figure
3a). Finally, the amplification efficiency (E) for each primer assay is calculated according to
the formula E=10
(-1/k)
. Since k (i.e. the slope of the trendline) for the ACTB primer assay
dilution curve is -3.3917, the amplification efficiency (E) for the ACTB primer assay is:

E = 10
(-1/-3.3917)
= 1.97

Using the same method, the E values for the P2RY1 and P2RY12 primer assays are found to
be 2.01 and 1.98.

8. Make a new mastermix (table 9) and quantify all genes of interest. It is recommended that at
least three biological replicates are used and that each gene is quantified at least in duplicate
reactions. In order to detect subtle changes in gene expression between samples and
controls, it is recommended to use at least eight biological replicates in each group.

Example: A total of 18 reactions are run for the quantification in duplicates of P2RY1, P2RY12
and ACTB using platelet cDNA from three individuals, resulting in the following Ct values for
platelet cDNA samples 1-3: P2RY1: 25.98, 25.32, 25.79; P2RY12: 22.7, 22.72, 22.92; ACTB:
18.58, 18.43, 18.6.

9. Calculate the expression of your genes of interest relative to your housekeeping gene using
the formula:

Relative expression (R) = (E
gene of interest
-Ct gene of interest
) / (E
housekeeping gene
-Ct housekeeping gene
)

Example: The expression of P2RY1and P2RY12 relative to ACTB in platelet cDNA sample 1 is:

R
P2RY1
= (2.01
-25.98
) / (1.97
-18.58
) = 0.0039

R
P2RY12
= (1.98
-22.7
) / (1.97
-18.58
) = 0.054

3.6 Preparation of RNA samples for platelet microarray analysis

There are many different microarray platforms available, and some of the more commonly used ones
are Illumina and Affymetrix. All microarray platforms have their pros and cons, and in most cases, the
choice of microarray platform is based on cost and which microarrays are available locally. Many
academic institutions now have core facilities specializing in microarray hybridizations and data
analysis, and it is highly recommended to let your local core facility handle all microarray
hybridizations.

If insufficient platelet RNA is available, it may also be necessary to either pool RNA from several
different platelet preparations(9-12) or to perform a double amplification of the platelet RNA (13).
Both methods have their pros and cons, and it is up to the researcher and the local microarray core
facility to find an optimal solution suitable for the intended project and the local conditions.

In order to validate gene expression results obtained by microarray, it is recommended that 5-10% of
the total amount of the RNA intended for microarray hybridization is set aside for cDNA synthesis
and qPCR validation of the microarray results.


4. Notes


1. The expected platelet yield may vary between donors. In general, dehydrated donors will
give a lower proportion of platelet rich plasma compared to the red blood cell fraction than
well hydrated donors. To increase platelet yield, it is therefore recommended that all blood
donors are well hydrated prior to blood donation. Various diseases and medications may also
affect the platelet count, which will influence the final platelet yield.
2. Depending on the type of magnet that is used (DynaMag-2 or DynaMag-15), either 1.5 or
2 mL tubes (DynaMag-2) or 15 mL tubes (DynaMag-15) are suitable for magnetic cell
depletion. For the sake of consistency, 2 mL tubes are stated throughout this protocol.
3. Self propagated blood flow is preferred in order to minimize platelet activation during the
blood collection process.
4. Platelet yield and gene expression profiles may show natural variations, in addition to
variations due to different disease states, medication and pregnancy.
5. TRIzol and chloroform are hazardous chemicals and should only be handled in a well
ventilated fume hood. Please read appropriate safety information before use.
6. RNA is very sensitive to degradation. Never touch any tubes, tips or surfaces that are to be
used for RNA isolation with bare hands. Avoid unnecessary talking and breathing towards the
samples while performing RNA extractions.
7. Always use filter tips during RNA isolation. Never reuse filter tips or use tips that have
accidentally touched any contaminated surface.
8. All volumes in this protocol are based on tubes containing platelets dissolved in 1 mL TRIzol.
If a platelet sample has been dissolved in more than 1 mL TRIzol, the sample will need to be
subdivided into several tubes of 1 mL TRIzol each during the RNA purification. The resulting
RNA pellets from the same donor can then be pooled into one tube during the RNA pellet
ethanol wash step (section 3.4, step 12).
9. In order to avoid accidental sample loss and contamination of the RNA samples, clear RNAse
free 1.5 mL tubes are preferred over large ultracentrifuge tubes for precipitation and
washing of the RNA samples
10. Chloroform easily drips out of 1000 L filter tips. To dispense chloroform, it is recommended
to use either 100 or 200 L tips.
11. A greater RNA yield can be achieved by precipitating the RNA at -20
o
C over night or at -80
o
C
for one hour in the presence of 10 g glycogen.
12. In our hands, reverse transcription using random hexamer primers has resulted in better
cDNA yields than cDNA generated using oligo-dT primers.
13. There are many different platforms for qPCR to choose from. We have had good success with
Qiagens QuantiFast SYBR Green kit and QuantiTect Primer assays using a Rotor-Gene 6000
PCR cycler. For an overview of other qPCR platforms such as TaqMan probes, please see
http://en.wikipedia.org/wiki/Real-time_PCR.
14. Several different housekeeping genes are available for relative real-time-PCR, including
GAPDH and ACTB that we have successfully used for platelet qPCR(1, 14).
15. For thermal cyclers other than Rotor-Gene 6000, see the QuantiFast SYBR Green PCR Kit
manual for optimal cycling conditions.
16. Scaling down the total qPCR reaction volume from 25 l to 10 l without reducing the
amount of cDNA added will result in a 60% reduction in QuantiFast SYBR Green cost and a
2.5 step lower Ct value for most qPCR reactions. Scaling down the reaction volume from 10
l to 5 l without reducing the amount of cDNA per reaction will further reduce the reagent
cost but will provide slightly higher Ct values compared to the 10 l reactions.
17. For most primer pairs, optimal linear quantification is achieved in the 17 to 30 Ct range (i.e.
17-30 PCR cycles). If a gene is detected later than 30 cycles, it may be difficult or impossible
to accurately quantify the gene of interest due to the low levels of template available for the
qPCR reaction. On the other hand, if a gene is detected earlier than 20 cycles, we
recommend that the template cDNA is diluted further in order to minimize waste of cDNA
template (table 10).


Figure Legends

Figure 1. Phase separation of TRIzol RNA samples after addition of chloroform. The clear aqueous
phase (top) contains the RNA, the white interphase (middle) contains DNA and the pink phenol phase
(bottom) contains protein. To avoid contamination of the RNA by DNA from the interphase it is
recommended that only 85% of the aqueous phase is transferred to a new tube.

Figure 2. Melt curve analysis following a real-time PCR run. A single peak (a) suggests that only one
amplification product is generated whereas no single peak is present in the non-specific melt curve
(b).

Figure 3. Primer efficiency plots. Following quantification of serially diluted cDNA template (see Table
7), an optimal qPCR assay (a) will be fit by a line with a slope (k) close to -3.3, which will translate into
a primer efficiency (E) of 2.0. b) A non-functional assay will give a slope which can be either greater
than, or less than the ideal slope of -3.3, resulting in a primer efficiency considerably different from
2.0.
Acknowledgments
The author would like to thank the Swedish Research Council and Prof. David Erlinge at Lund
University, Sweden for their support.


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Table 1. Thermal cycling conditions for reverse transcription using the TaqMan reverse
transcription kit.

Table 2. Preparation of master mix for cDNA synthesis. All volumes are in l.

Table 3. Rotor-Gene 6000 cycling conditions for qPCR using the QuantiFast SYBR Green PCR
Kit.

Table 4. Preparation of master mix solution for pilot qPCR quantification using the
QuantiFast SYBR Green PCR Kit.

Table 5. Preparation of master mix for determination of primer assay efficiency.

Table 6. Calculations of required amounts of cDNA for determination of primer amplification
efficiency for the genes P2RY1, P2RY12 AND ACTB.

Table 7. Volumes required for serial dilutions of cDNA for primer amplification efficiency
determination.

Table 8. Calculation of average Ct and dilution factor log values for the dilution curve of
ACTB.

Table 9. Preparation of mastermix for the quantification of one gene in duplicate reactions in
at least three biological replicates, resulting in a master mix for 7 reactions ( 3 x 2 + 1 =7
reactions).

Table 10. Potential of cDNA dilution in order to economize cDNA without compromising
qPCR quality and accuracy. If the pilot qPCR returns Ct values lower than 20 to 25 cycles, it is
possible to dilute the template cDNA without compromising qPCR quality and accuracy.

Table 1.

Temp C Time (minutes)
25 10
48 30
95 5
4 Eternity

Table 2.

Reaction volume 25 L
Make for number of reactions 1x
10x TaqMan RT-buffer 2.50
25mM MgCl
2
5.50
10 mM dNTP 5.00
Random hexamers 1.25
RNase inhibitor 0.50
MultiScribe Transcriptase 0.65
Master mix to each tube 15.40
RNA to each tube (60-2000 ng) 9.60

Table 3.

Step Temp (C) Time (sec) Flourescence acquisition
1 Initial denaturation 95 300 None
2 Cycling Denaturation 95 10 None
3 Cycling Annealing/Extension 60 30 Sybr/FAM
4 Repeat step 2-3 40 cycles
5 Melt curve 65-95* 5-30* Sybr/FAM
*Rising by 1 C each step, wait for 30 sec on first step, then wait for 5 sec for each step afterwards.

Table 4.

Number of 10 L assays 1 x 10 l 3.5 x 10 l
Mastermix 5 17.5
Primer assay mix 0 0
Diluted cDNA 1 3.5
Volume mastermix added to each qPCR reaction 6 6
Volume QuantiTect Primer assay diluted 1 in 4 added
to each qPCR reaction 4 4


Table 5.

Number of 10 L assays 1 x 10 l 17.5 x 10 l
Mastermix 5 87.5
QuantiTect Primer assay 1 17.5
Volume mastermix added to each qPCR reaction 6 6
Volume diluted cDNA added to each qPCR reaction 4 4

Table 6.

Number of primer assays to test +1 4
Technical replicates 3
Diluted cDNA needed for each dilution factor step (number of assays x technical
replicates) 12
Extra water to add so that 4 l diluted cDNA can be added per assay* 36
*It is easier to accurately pipette 4L than 1 L, so extra water is added to the diluted cDNA
to allow 4 L to be added to each reaction tube.


Table 7.

Final cDNA
dilution factor Volume cDNA and water added to each dilution step
1 6 L of undiluted cDNA stock + 90 L water
2 48 L water + 48 L cDNA from the above dilution step
4 48 L water + 48 L cDNA from the above dilution step
8 48 L water + 48 L cDNA from the above dilution step
16 48 L water + 48 L cDNA from the above dilution step


Table 8.

Dilution factor Log dilution factor Ct repl. 1 Ct repl. 2 Ct repl. 3 Average Ct
1 0.00 17.19 17.29 17.29 17.26
2 -0.30 18.38 18.42 18.20 18.33
4 -0.60 19.20 19.33 19.34 19.29
8 -0.90 20.30 20.37 20.35 20.34
16 -1.20 21.31 21.39 21.38 21.36


Table 9.

Number of 10 L assays 1 x 10 l 7 x 10 l
Mastermix 5 35
Volume undiluted QuantiTect Primer assay 1 7
Volume mastermix added to each qPCR reaction 6 6
Volume diluted cDNA added to each qPCR reaction 4 4

Table 10.

detection Ct value
(qPCR cycle number)
cDNA dilution factor
to reach Ct 20
cDNA dilution factor
to reach Ct 25
15 32 1024
16 16 512
17 8 256
18 4 128
19 2 64
20 1 32
21

16
22

8
23

4
24

2
25

1


Interphase with DNA
Aqueous phase with RNA
Phenol phase with protein
Transfer only 85%
of aqueous phase
to avoid DNA
contamination
Figure 1
Specific melt curve
Figure 2a
Non-specific melt curve
Figure 2b
Figure 3.
a) b)
E=10
(-1/3.39)
=1.97 E=10
(-1/1.32)
=5.70
Ideal primer pair
Poorly performing primer pair