The following procedures describe the measurement of the ratio of the alleles of a SNP (dbSNP accession number rs8130833) on PLAC4 mRNA (NCBI reference sequence NM_182832) in maternal plasma.
1) Collect at least 3.5 mL of blood into EDTA tubes. Store the blood samples at 4 °C for a maximum of 6 h if processing procedures cannot take place immediately.
CRITICAL STEP If the blood samples are not processed promptly or within 6 h while stored at 4 °C, concentration of background RNA may increase and may interfere with the analysis of the targeted transcript4.
2) Centrifuge the whole blood at 1,600g for 10 min at 4 °C. Transfer plasma into clean polypropylene tubes carefully, so as not to disturb the cellular layer.
3) Recentrifuge the plasma at 16,000g for 10 min at 4 °C. Carefully transfer the supernatant into clean polypropylene tubes without disturbing the cell pellet. Discard the pellet.
4) Add 1.2 mL of Trizol LS reagent per 0.4 mL of plasma in a clean 2 mL polypropylene tube. Vortex vigorously. Store the plasma-Trizol LS mixture at -80 °C until RNA extraction.
CRITICAL STEP Inadequate vortexing would lead to a lower yield of plasma RNA.
PAUSE POINT RNA in plasma-Trizol LS mixture is stable at -80 °C for up to three years5.
Plasma RNA extraction
Perform all procedures and keep all reagents at room temperature unless otherwise specified.
CRITICAL STEP To minimize the introduction of RNase during the extraction, clean the laboratory bench and the equipment with RNase Away (Molecular BioProducts, cat. no. 7000) before each extraction. Use sterile RNase-free plasticware for the procedures.
5) Thaw 6.4 mL of the plasma-Trizol LS mixture prepared in step 4.
6) For every 1.6 mL of the plasma-Trizol LS mixture, add 320 µL of chloroform. Shake the tubes vigorously by hand. Centrifuge the mixture at 12,000g for 15 min at 4 °C.
7) Transfer the upper aqueous layer into new tubes. The volume of the aqueous layer is approximately 850 µL for each 1.6 mL of the plasma-Trizol-LS mixture. Add 457 µL of absolute ethanol per 850 µL of the aqueous layer and mix by inverting the tubes.
8) Apply 700 µL of the mixture into an RNeasy minicolumn. Centrifuge at 9300g for 15 s. Discard the flow-through.
9) Apply another 700 µL of the mixture into the same extraction column. Repeat step 8 for seven more times until all mixture is loaded.
10) Add 700 µL of Buffer RW1 to the column. Centrifuge at 9300g for 1 min. Discard the flow-through.
11) Add 500 µL of Buffer RPE to the column. Centrifuge at 9300g for 15 s. Discard the flow-through.
12) Add another 500 µL of Buffer RPE to the column. Centrifuge at 16,000g for 2 min. Discard the flow-through.
13) Centrifuge the column at 16,000g for 1 min.
14) Place the column in a new 1.5 mL tube. To elute the RNA, add 48 µL of RNase-free water into the column and incubate for 5 min at room temperature. Centrifuge the column at 16,000g for 1 min.
15) Apply the eluate into the same column and incubate for another 5 min at room temperature. Centrifuge the column at 16,000g for 1 min.
PAUSE POINT Store the extracted RNA at -80 °C if DNase I digestion cannot be carried out immediately.
16)Perform DNase I digestion
a)Add 4.8 µL each of 10x DNase I Reaction Buffer (provided with the DNase I) and DNase I to 48 µL of plasma RNA on ice. Mix by pipetting. Incubate the reaction for 15 min at room temperature.
b)Add 4.8 µL of EDTA (provided with the DNase I). Incubate the reaction for 10 min at 65 °C and then place the tubes on ice.
CRITICAL STEP DNase I digestion is performed to eliminate any contaminating genomic DNA, which if present, could adversely affect the observed allelic ratio.
17) Store the RNA at -80 °C
Real-time quantitative RT-PCR (QRT-PCR)
Plasma RNA sample is firstly quantified with QRT-PCR2 for PLAC4 mRNA. This is to ensure the amount of RNA in the sample is sufficient for the subsequent RNA allelic ratio determination3.
18) Prepare a calibration curve by serially diluting the synthetic DNA oligonucleotide into the following concentrations: 2.5 x 106, 2.5 x 105, 2.5 x 104, 2.5 x 103, 2.5 x 102, 25 and 2.5 copies/µL.
CRITICAL STEP Preparation of the calibration curve is a step which has a high contamination risk since each of the constituent dilutions contains a very high concentration of short synthetic oligonucleotide specifying the amplicon. Thus, it is crucial to prepare the calibration curve in an area that is physically separated from the PCR preparation site.
19) Prepare QRT-PCR reaction master mix with the following components (for one reaction):
5 µL of 5x TaqMan EZ Buffer
3 µL of manganese acetate
0.75 µL of dATP
0.75 µL of dCTP
0.75 µL of dGTP
0.75 µL of dUTP
1 µL of forward QRT-PCR primer
1 µL of reverse QRT-PCR primer
0.5 µL of TaqMan MGB probe
1 µL of rTth DNA Polymerase
0.25 µL of AmpErase uracil N-glycosylase (UNG)
5.25 µL of water
20) Aliquot 20 µL of the master mix into each well.
21) Perform duplicated reactions for each sample, no template control (NTC) and calibration curve. To the sample wells, add 5 µL of RNA sample. To the NTC wells, add 5 µL of RNase free water.
22) Cover the wells with optical caps.
CRITICAL STEP To prevent calibration curve-associated contamination, cap both the samples and the NTC wells immediately before adding the calibration curve.
23) For the calibration curve prepared in step 18, add 5 µL of the seven concentrations of synthetic oligonucleotide individually into the calibration wells. Cover the wells with optical caps.
24) Perform QRT-PCR as follows:
50 °C for 2 min
60 °C for 30 min
95 °C for 5 min
45 cycles of 95 °C for 15 s, 60 °C for 1 min
25) Calculate RNA concentration according to the following equation:
C = Q x (VRNA / VPlasma)
in which C represents PLAC4 mRNA concentration in plasma (copies/mL), Q represents PLAC4 mRNA concentration of the extracted RNA (copies/µL), which is determined by the sequence detector, VRNA represents total volume of the DNase-treated RNA after extraction (62.4 µL), VPlasma represents volume of plasma used for extraction (1.6 mL).
For the NTC, no amplification signal should be observed.
CRITICAL STEP If the RNA concentration of a particular sample is very low, extra care should be taken when interpreting the result of that sample in the subsequent RNA allelic ratio analysis. It has been shown that when the starting RNA amount of a sample is very low, the chance of inaccurate allelic ratio measurement might increase3.
26) Add 4.8 µL of gene-specific primer and 9.6 µL of dNTP mixture to 48 µL of the DNase-treated plasma RNA prepared in step 16. Incubate the mixture at 65 °C for 5 min and then place on ice.
27) Add 19.2 µL of 5x cDNA Synthesis Buffer (provided with the ThermoScript enzyme), 4.8 µL each of dithiotreitol (DTT) (provided with the ThermoScript enzyme), RNaseOUT and the ThermoScript enzyme into the reaction. Immediately place the mixture in a thermocycler that has been pre-heated at 55 °C.
CRITICAL STEP Minimize the handling time between ice and 55 °C incubation to reduce RNA secondary structure formation and non-specific priming by the ThermoScript enzyme.
28) Incubate the reaction at 55 °C for 60 min, follow by 85 °C at 5 min.
PAUSE POINT Store the cDNA at -20 °C if PCR amplification cannot be carried out immediately.
29) Prepare PCR reaction with the following components (for one reaction).
12 µL of 10x PCR Buffer (containing 15 mM MgCl2)
2 µL of dNTP mixture
4 µL of forward PCR primer
4 µL of reverse PCR primer
0.8 µL of HotStar Taq polymerase
77.2µL of water
100 µL of synthesized cDNA from step 28
(Since the cDNA product contains significant amount of magnesium salts, and constitutes half of the PCR reaction volume, no additional MgCl2 and less 10x PCR buffer should be added in order to maintain an optimal salt concentration for the PCR reaction)
30) To control for any PCR product contamination, prepare a NTC tube according to the components in step 29. Replace the cDNA with 100 µL of water.
31) Perform PCR as follows:
95 °C for 7 min
55 cycles of 95 °C for 40 s, 56 °C for 1 min, and 72 °C for 1 min
72 °C for 3 min
PAUSE POINT Store the PCR product at -20 °C if base extension reaction cannot be carried out immediately. The PCR product can be stored for at most three months. Prolonged storage may adversely affect the RNA allelic ratio.
Base extension reaction
32) Dephosphorylate any remaining dNTPs in the PCR product with shrimp alkaline phosphatase (SAP). This is to prevent their incorporation in the subsequent base extension assay.
a)Add 0.34 µL of hME buffer and 0.6 µL of SAP to 25 µL of the PCR product and mix thoroughly.
b)Incubate at 37 °C for 40 min, followed by 85 °C for 5 min.
33) Prepare base extension reaction with the following components (for one reaction):
0.4 µL of ddATP/ddCTP/ddTTP/dGTP mixture
1.68 µL of extension primer
0.036 µL of Thermosequenase
6.884 µL of water
5 µL of SAP-treated PCR product from step 32
34) Perform the reaction as follows:
94 °C for 2 min
100 cycles of 94 °C for 5 s, 52 °C for 5 s, and 72 °C for 5 s
PAUSE POINT Store the base extension product at -20 °C if MALDI-TOF MS analysis cannot be carried out immediately. Avoid storing the base extension product for more than two weeks as prolonged storage may adversely affect the RNA allelic ratio.
MALDI-TOF MS analysis
35) Remove cations in the base extension reaction product using SpectroCLEAN resin.
a)Add 24 µL of water and 12 mg of resin to the reaction product.
b)Place the mixture in a rotator for 30 min at room temperature.
36) Centrifuge the mixture at 361g for 5 min.
37) Dispense the final reaction solution onto a SpectroCHIP by SpectroPoint nanodispenser. Refer to “MassArray Nanodispenser S User’s Guide” (Sequenom) for instructions.
38) Acquire mass spectrometric data by a MassARRAY Analyzer Compact Mass Spectrometer. Refer to “MassARRAY Analyzer Compact User’s Guide” (Sequenom) for instructions.