REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION (Q-PCR)

 

Attila Szanto

 

University of Debrecen, Medical and Health Science Center, Department of Biochemistry and Molecular Biology, Life Science Building, Room 3-211, Egyetem ter 1, Debrecen, H-4010, Hungary, Tel.: +36 52 416432, Fax: +36 52 314989

[email protected]

 

 

INTRODUCTION TO THE PROCEDURE

 

Real-time quantitative polymerase chain reaction (Q-PCR) is the most sensitive method for detection and quantification of RNA or DNA molecules. It is a complex method that requires expertise in PCR assay development and optimization. Nevertheless, it is a powerful method with high specificity, sensitivity and reproducibility that can be used in high-throughput studies. Basically, Q-PCR is a fluorescence-based kinetic study where fluorescent signal produced by the proceeding amplification of a specific template is determined in every PCR cycle. The real time determination of the fluorescent signal makes it possible to use the log phase of the PCR for quantification, when the signal correlates with the initial number of template molecules, and at the same time provides high sensitivity when comparing small differences. Another advantage of the technique is that molecules with low-abundancy can also be quantified precisely, allowing the analysis of limiting amounts of cells or tissues.

 

Reverse transcription

 

For the analysis of the transcriptome we need quantification of the expressed RNA molecules. There are five commonly used methods for this: Northern-blotting, RNase protection assays, in situ hybridization, microarrays and reverse transcription (RT)-PCR. Among these real time RT-Q-PCR is the most sensitive and suitable method for quantification. To analyze RNA molecules with Q-PCR, they need to be transcribed into cDNA molecules by reverse transcriptase. This can be primed by different methods: gene-specific primers, random primers or oligo-dT primers.

 

Quantification of cDNA/DNA molecules

 

Quantitative information is generated from fluorescent signal that correlates with the initial copy number of cDNA or DNA molecules. This signal can be generated from non-specific binding of a fluorescent dye to the amplified nucleic acid (e.g. SYBR Green I) or from sequence specific, fluorescently labeled oligonucleotides, also called probes (e.g. Taqman probes). Both methods can quantify the mRNA/DNA copy numbers of the gene-of-interest, either in an absolute manner when using standard curves (serial dilution of an amplicon with know copy numbers), or in a relative manner when comparing to a specific sample. In both cases normalization to A) a consitutively expressed and not regulated mRNA or B) a gene with stable genomic copy number is essential.

 

 

 

OUTLINE OF THE EXPERIMENTS

 

 

In these experiments we will analyze the effects of DMSO priming and 9-cis retinoic acid treatment of CDM1 cells on different levels. Previous experiments are analyzed with one common method, Q-PCR in the following manner:

 

• Practical 8 – Quantification of ChIP DNA
  Epigenetic modifications are analyzed from the chromatin immunoprecipitation experiment (Chapter III, Practical 6).
  Each group will analyze their four samples (Input, No-antibody control – NAB, Histone 4 polyacetylation – H4Ac and Histone 3 Lysine 4 Methylation – H3K4Met) by measuring four DNA fragments (transglutaminase 2 core promoter, transglutaminase 2 HR1 enhancer region, CD38 Retinoic Acid Receptor responsive element and 36B4 gene).

 

• Practical 9 – Quantification of mRNA
  Gene expression changes are analyzed by measuring retinoic acid induced target gene expression in the same RNA samples used in the microarray experiment (Chapter I, Practical 1).
  Each group will analyze their sample by measuring the expression of two genes-of-interest (both are retinoid receptor target genes: transglutaminase 2 and CD38) and two normalizer genes.

 

• Practical 10 – Quantification of miRNA
  Expression of miRNAs are also analyzed in RNA samples derived from the same cells (Chapter I, Practical 2).
  Each group will analyze their sample by measuring the abundance of two miRNAs and one normalizer.

 

REAL-TIME QUANTITATIVE PCR ANALYSIS - PRACTICALS

 

 

Practical 8 – Quantification of ChIP DNA

 

Each group will analyze four samples:

            Input

            No Antibody Control (NAB)

            H4Ac

            H3K4Met

 

Four genes will be measured from every sample:

 

                        Transglutaminase 2 core promoter (Core)

                        Transglutaminase enhancer HR1 (HR1)

                        CD38 Retinoic Acid Receptor Response Element (CD38)

                        36B4 (intron-less gene for control)

 

Q-PCR analysis will be performed in 384 well plates with a pipetting robot.

 

 

 

Practical 8A: Real-time PCR with TaqMan probes

 

Checklist:

            Samples in strips:       Input

                                                NAB

                                                H4Ac

                                                H3K4Met

            2x Taqman Master Mix

            Primers and Probes

 

• Thaw the strips with DNA samples from ChIP experiment (Practical 3).

 

• Mix gently and place on ice.

 

DNA samples will be measured in triplicates. Six groups will use one plate. Prepare DNA samples and PCR Master mixes according to Figure 1. Note, that six groups prepare four PCR Master mixes.

 

 

 

 

 

 

• Mix the following components for 100 reactions in 1.5 ml Eppendorf tubes for four genes (Core, HR1, CD38 and 36B4, respectively) on ice.

 

 

                                                            (For one reaction)                   For 100 reactions

 

2x Taqman Master Mix                       5 ml                                          500 ml 

Forward primer (100 mM)                  0.0375 ml                                 3.75 ml

Reverse primer (100 mM)                   0.0375 ml                                 3.75 ml

Probe (20 mM)                                                0.0625 ml                                 6.25 ml

Final Volume (approximately)            5 ml                                          500 ml

 

 

• Mix

 

• Pipette 60 ml PCR Master mix into each well of an 8-well strip and place it as indicated in Figure 1.

 

When both the samples and the PCR master mixes are located in the correct position the robot will be started.

 

When the plate is finished cover it with adhesive optical cover and spin it in the plate centrifuge. Then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

           

 

 

PCR cycle:      1. 95^oC                        10 min

                                    2. 95^oC                        15 sec

                                    3. 60^oC                        60 sec

                                    Repeat Steps 2 and 3 40 times

 

• Analyze Q-PCR results with SDS software.
• Export Results Table.
• Analyze data in Excel.

Practical 9 – Quantification of mRNA

 

 

Practical 9A: Establishing Standard Curves

 

Checklist:

            Transglutaminase amplicon (6e7)

            Marked Eppendorf tubes

            Yeast tRNA

            2x Taqman Master Mix

            Primers and Probe

            Nuclease-free water

 

1. Make a serial dilution of Transglutaminase 2 amplicon.

• Thaw Yeast Transfer RNA (tRNA) Solution (100 ng/ml).
• Take 7 marked Eppendorf tubes:

            6e7

            6e6

            6e5

            6e4

            6e3

            6e2

            6e1

• Pipette 18 ml tRNA into each tubes.
• Thaw Tg amplicon (6e7).
• Vortex for 30 sec
• Spin down in Eppendorf centrifuge (15 sec, maximum speed, room temperature)
• Pipette 2 ml of Tg amplicon to the 6e6 tube to make a 10-fold dilution.
• Vortex for 30 sec
• Spin in Eppendorf centrifuge (15 sec, maximum speed)

 

Repeat these steps with the other tubes (6e5 to 6e1), pipetting Tg amplicon from the dilution made in the previous step to obtain all the 7 log dilution series of the amplicon.

 

2. Prepare PCR Master mix for Transglutaminase for 30 reactions:

• Mix the following components in a 1.5 ml Eppendorf tube on ice:

 

                                                            (For one reaction)                   For 30 reactions

 

2x Taqman Master Mix                       10 ml                                        300 ml 

Forward primer (100 mM)                  0.075 ml                                   2.25 ml

Reverse primer (100 mM)                   0.075 ml                                   2.25 ml

Probe (20 mM)                                                0.125 ml                                   3.75 ml

Nuclease-free water                             7.725 ml                                   231.75 ml

Final Volume                                      18 ml

 

• Mix by vortexing
• Spin down briefly

 

3. Fill up the 96-well PCR plate:

 

Samples will be measured in triplicates. Similarly, No Template Control (NTC) will be measured in triplicates as indicated in Figure 2. Note that 4 groups will use one plate.

 

• Pipette 18 ml PCR Master mix into the indicated wells of a 96 well plate.
• Pipette 2 ml nuclease-free water into the first 3 wells (i.e. A1-3) and continue with the diluted amplicon samples as indicated in Figure 2 (i.e. 6e1 dilution into B1-3 wells).

 

 

 

• When the plate is finished cover it with adhesive optical cover and spin it in plate centrifuge then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

 

            PCR cycle:      1. 95^oC            10 min

                                    2. 95^oC            12 s

                                    3. 60^oC            30 s

                                    Repeat Steps 2 and 3 40 times

 

• Analyze Q-PCR results with SDS software.

Practical 9B: Reverse Transcription

 

The same RNA used for the microarray experiments will be analyzed in this experiment. Note that a portion of the RNA prepared with the Qiagen RNeasy kit was DNase-treated prior to this experiment.

 

Checklist:

            RNA sample from Practical 1

            5x First Strand Buffer

            dNTP (2.5 mM)

            DTT (0.1 M)

            Nuclease-free water

            Random primer

            Reverse Transcriptase will be provided by the assistance

 

• Thaw RNA samples (Qiagen preps).
• Mix and place on ice.

 

• Prepare two Master mixes in thin walled 0.2 ml PCR tubes on ice:

 

For one reaction

RT                               No-RT

 

5x First Strand Buffer                                     8 ml                              3 ml

dNTP (2.5 mM)                                              8 ml                              3 ml

DTT (0.1 M)                                                   4 ml                              1.5 ml

Random Primer (1 mg/ml)                                1.2 ml                           0.45 ml

Reverse Transcriptase (200 U/ml)                   0.4 ml                           -

Final Volume                                                  21.6 ml                         7.95 ml

 

Add DNase-treated total RNA (150 ng/ml)     18.4 ml                         6.9 ml

Nuclease-free water                                         -                                   0.15 ml

Final Volume                                                  40 ml                            15 ml

 

• Put tubes into PCR machine

 

            PCR cycle:      25^oC    10 min

                                    42^oC    2 h

                                    72^oC    5 min

                                    4^oC      hold

 

cDNA samples can be stored at -20^oC.

Practical 9C: Real-time PCR with TaqMan probes

 

 

Every group will measure four genes: Cyclophylin

                                                                        36B4

                                                                        Transglutaminase 2 (Tgm2)

                                                                        CD38

 

Checklist:

            cDNA samples (RT and No-RT) from Practical 9B

            2x TaqMan Master Mix

            Nuclease-free water

            Forward, reverse primers and Probes

 

• Thaw cDNA samples (RT and No-RT).
• Dilute samples 5-fold with Nuclease-free water
• Mix

 

1. Prepare PCR Master mixes for five reactions (one for each gene being measured)

 

·      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

 

                                                            (For one reaction)                   For 5 reactions

 

2x Taqman Master Mix                       10 ml                                        50 ml   

Forward primer (10 mM)                    0.75 ml                                     3.75 ml

Reverse primer (10 mM)                     0.75 ml                                     3.75 ml

Probe (10 mM)                                                0.25 ml                                     1.25 ml

Nuclease-free water                             3.25 ml                                     16.25 ml

Final Volume                                      15 ml

 

·      Mix by vortexing, then spin down briefly in centrifuge

 

2. Fill up the 96-well PCR plate:

RT samples will be measured in triplicates while No-RT control will be measured in a single well. Six groups will use one plate.

 

·      Pipette 15 ml PCR Master mix into 4 wells of a 96-well plate as indicated in Figure 3.

·      Pipette 5 ml of cDNA (RT) into the first 3 wells and 5 ml sample (No-RT) into the 4^th well.

·      Repeat these with the other genes as indicated in Figure 3.

 

·      When the plate is finished cover it with adhesive optical cover and spin it in plate centrifuge then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

            PCR cycle:      1. 95^oC                        10 min

                                    2. 95^oC                        12 sec

                                    3. 60^oC                        30 sec

                                    Repeat Steps 2 and 3 40 times

 

• Analyze Q-PCR results with SDS software.
• Export Results Table.
• Analyze data in Excel.

 

Alternative protocol for the PCR, if not using a commercial master mix:

 

                                                            For one reaction

10x PCR Buffer                                   2 ml

MgCl₂ (25 mM)                                   2.4 ml

dNTP (2.5 mM)                                   1 ml

Forward primer (100 mM)                 0.75 ml

Reverse primer (100 mM)                   0.75 ml

Probe (20 mM)                                    0.125 ml

Taq polymerase (5 U/ml)                    0.125 ml

ROX reference (50x)                           0.4 ml

Nuclease-free water                            7.45 ml

Final Volume                                       15 ml

 

cDNA                                                  5 ml

Final Volume                                       20 ml

Practical 10 – Quantification of miRNA

 

 

Practical 10A: Reverse Transcription

 

We are using High Capacity cDNA Archive Kit (Applied Biosystems) to transcribe miRNAs from Trizol-purified total RNA samples. Note that Qiagen preps cannot be used for miRNA analysis because small RNA molecules are lost during that procedure while they are kept during Trizol purification.

 

The following miRNAs will be measured:      hsa-miR-34a

                                                                        hsa-let-7i

                                                                        hsa-miR-338

                                                                        hsa-let-7a

RNU43 (U43 small nucleolar RNA)

 

Each group will measure two miRNAs and one normalizer small nucleolar RNA:

 

            Group 1, 4, 7 and 10:  hsa-miR-34a

                                                hsa-let-7a

                                                RNU43

            Group 2, 5, 8 and 11:  hsa-miR-7i

                                                hsa-let-7a

                                                RNU43

            Group 3, 6, 9 and 12:  hsa-miR-338

                                                hsa-let-7a

                                                RNU43

 

Checklist:

            Trizol-isolated RNA samples from Practical 2

            10xRT Buffer

            dNTP (10 mM)

            Nuclease-free water

            Reverse Transcriptase, RNAsin and 5xRT Primer will be provided by the     assistance

 

• Thaw RNA samples (Trizol preps).

 

• Mix and place on ice.

 

 

 

 

 

 

 

 

 

1. Prepare RT master mix for four reactions in a 1.5 ml Eppendorf tube on ice:

 

·      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

 

 

RT 1-3

(For one reaction)                   For 4 reactions

 

10xRT Buffer                                      1.2 ml                                       4.8 ml

dNTP (10 mM)                                   1.2 ml                                       4.8 ml

Reverse Transcriptase (50 U/ml)         0.8 ml                                       3.2 ml

RNAsin (40 U/ml)                               0.15 ml                                     0.6 ml

Nuclease-free water                             4.25 ml                                     17 ml

 

Final Volume                                      7.6 ml                                       30.4 ml

 

• Mix and pipette 7.6 ml master mix into 3 PCR tubes: RT 1, 2 and 3.
• Add 2.4 ml of the indicated 5x Reverse primer (1-3) to each:
• Add 2 ml of total RNA (20 ng/ml) to each tube

That will give you a final volume of 12 ml.

 

• Mix
• Put tubes on ice

 

2. Prepare the following No-RT master mix for four reactions in a 1.5 ml Eppendorf tube on ice.

 

·               Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

No-RT 1-3

(For one reaction)                   For 4 reactions

 

10x RT Buffer                                     1.2 ml                                       4.8 ml

dNTP (10 mM)                                   1.2 ml                                       4.8 ml

RNAsin (40 U/ml)                               0.15 ml                                     0.6 ml

Nuclease-free water                             5.05 ml                                     20.2 ml

 

Final Volume                                      7.6 ml                                       30.4 ml

 

• Mix and pipette 7.6 ml master mix into 3 PCR tubes: No-RT 1, 2 and 3.
• Add 2.4 ml of the indicated 5x Reverse primer (1-3) to each tube
• Add 2 ml of total RNA (20 ng/ml) to each tube

That will give you a final volume of 12 ml.

• Mix tubes gently
• Put tubes into PCR

            PCR cycle:      16^oC    30 min

                                    42^oC    30 min

                                    85^oC    5 min

                                    4^oC      hold

Tubes can be stored at -20^oC.

Practical 10B: Real-time PCR with TaqMan probes

 

Checklist:

            Reverse transcribed miRNA samples from Practical 10A

            2x TaqMan Master Mix

            Nuclease-free water

            10x TaqMan miRNA assays will be provided by the assistance

 

• Thaw cDNA samples (RT1-3 and No-RT1-3)
• Mix

 

RT samples will be measured in triplicates while No-RT control will be measured in a single well. 6 groups will use one plate. See Figure 4.

 

1. Prepare PCR Master mixes 1, 2, 3 (one for each miRNA being measured), respectively.

 

·      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

                                                            (For one reaction)                   For 5 reactions

 

2x Taqman Master Mix                       7.5 ml                                       37.5 ml

10x Taqman miRNA assay                 1.5 ml                                       7.5 ml

Nuclease-free water                             4.5 ml                                       22.5 ml

 

Final Volume                                      13.5 ml                                     67.5 ml

 

·      Mix

 

 

 

·      Pipette 13.5 ml PCR Master mix into 4 wells of a 96 well plate as indicated in Figure 3.

·      Pipette 1.5 ml of RT Product (RT) into the first 3 wells and 1.5 ml RT Product (No-RT) into the 4^th well as indicated in Figure 4.

·      When the plate is finished cover it with adhesive optical cover and spin it in plate centrifuge then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

            PCR cycle:      1. 95^oC            10 min

                                    2. 95^oC            15 s

                                    3. 60^oC            60 s

                                    Repeat Steps 2 and 3 40 times

 

• Analyze Q-PCR results with SDS software.
• Export Results Table.
• Analyze data in Excel.

 

Tips and tricks

 

• When diluting amplicon always use carrier in the solution. Transfer RNA is ideal. At low concentrations the few molecules are easily attached to the surface of the plastic tube when water is used and the probability of degradation is higher.

 

• When working with concentrated amplicon (>1fmol/ml) use other pipette set than during performing RT or PCR to avoid contamination. Different lab is optimal.

 

• In vitro transcribed synthetic RNA can be also used as amplicon. In this case radioactive labeling should be used for quantification.

 

• When many transcripts are measured from the same sample using random primers for RT is more economical than other methods. But if only a few transcripts are measured specific reverse primer can be also used. Always use the same RT method for every gene analyzed. When low quality RNA is analyzed do not use oligo dT.

 

• While the general amplicon size is below 100 bp the quality of RNA is not critical in Q-PCR.

 

• In specific reverse primed RT 30 min is enough while in random primed reactions 2h is recommended.

 

• Use triplicates and No-RT control in Q-PCR measurements.

 

• Taqman assay is not that expensive and is recommended. Optimization of a SYB Green assay would need several primer group combinations and master mix and could result in a relatively early non-specific signal in the NTCs.

 

• Do not use Qiagen purified RNA for miRNA quantification.

 

Reagents

 

2x SYBR Green Master Mix – Diagenode – GMO-SG2x-A300

2x Taqman Master Mix – Diagenode – GMO-MM2x-A300

Yeast Transfer RNA 25 mg – Invitrogen – 15401-011

Primers – Sigma-Genosys

SuperScript II RNase H-Reverse Transcriptase, 10000U, 200U/ml – Invitrogen – 18064-014

5xFirst Strand Buffer – Invitrogen – Y00146

DTT, 0.1 mM, 500 ml – Invitrogen – Y00147

Random Primer, 300 mg, 3 mg/ml – Invitrogen – 48190-011

20x Reference Dye, 500 ml – Invitrogen – 54881

dATP, 100 mM, 25 mmol – Fermentas – R0141

dCTP, 100 mM, 25 mmol – Fermentas – R0151

dGTP, 100 mM, 25 mmol – Fermentas – R0161

dTTP, 100 mM, 25 mmol – Fermentas – R0171

RiboLock Ribonuclease Inhibitor, 40 U/ml, 2500U – Fermentas – EO0381

Taq DNA Polymerase, 5 U/ml, 500U – Fermentas – EP0402

miRNA Assays – Applied Biosystems

cDNA Archive Kit, 200 rxn, 100 ml – Applied Biosystems – 4322171