RealTimePCR-Protocol-miRNA-SCALONMC
Marcela Scalon, Christine Souza Martins, Gabriel Ginani Ferreira, Franciele Schlemmer, Ricardo Titze de Almeida, Giane Regina Paludo
Abstract
This protocol is intended as a guideline to perform the Real Time PCR (qPCR or rtPCR) procedure to detect and quantify miRNAs. This method use TaqMan® MicroRNA Assays and Applied Biosystems real-time PCR instruments. The assays are preformulated primer and probe sets designed to detect and quantify miRNAs, and can detect and quantify small RNA in 1 to 10 ng of total RNA with a dynamic range of greater than six logs. This protocol works on detecting miRNA from samples that were purified from plasma and serum. The amplified products of the RT procedure must be stored − 15 to − 25 °C, if not used immediately for rtPCR. The aim of this protocol is to use a sequence- specific TaqMan® assay to accurately detect miRNAs.
Before start
Thoroughly mix the TaqMan® Fast Advanced Master Mix by swirling the bottle.
Thaw frozen TaqMan® assays by placing them on ice. When thawed, resuspend the assays by vortexing, then briefly centrifuge.
Thaw frozen samples by placing them on ice. When thawed, resuspend the samples by briefly vortexing, then briefly centrifuge.
Steps
Prepare the real-time PCR (qPCR or rtPCR) reaction mix
Determine the total number of PCR reactions to perform. On each reaction plate include:
• A miRNA assay for each cDNA sample
• Control assays
• No template controls (NTCs) for each assay on the plate
Notes:
-
It is possible to run multiple assays on one reaction plate. Include controls for each assay.
-
Perform 3 replicates of each reaction
Per the table below, calculate the total volume required for each reaction component.
Note: Always include extra volume to compensate for the volume loss that occurs during pipetting.
| A | B |
|---|---|
| Component | Volume (μL) for 1 reaction |
| TaqMan® Fast Advanced Master Mix | 10.00 μL |
| Primer TaqMan® target or control (20✕) | 1.00 μL |
| Nuclease-free Water | 7.00 μL |
| Total volume per reaction | 18.00 μL |
Label the microtubes (0.6-mL or 1.5-mL).
Add all the components at the calculated quantities.
Mix gently, then centrifuge to bring the solution to the bottom of the tube and eliminate air bubbles.
Prepare de PCR reaction plate
Transfer 18.00 μL of each PCR reaction mix to each well of an optical reaction plate (MicroAmp® Fast Optical 96-Well Reaction Plate).
Add the cDNA or Nuclease-free Water, to the respective well of the plate, as follows:
| A | B |
|---|---|
| Component | Volume (μL) for 1 reaction |
| cDNA or Nuclease-free Water (for NTC) | 2.00 μL |
| Total volume per well | 20.00 μL |
Cover the reaction plate with an optical adhesive film (MicroAmp® Optical Adhesive Film).
Centrifuge the plate briefly to spin down the contents and eliminate air bubbles.
Run the rtPCR reaction plate
In the system software, open the plate document or experiment that corresponds to the reaction plate.
Load the reaction plate into the real-time PCR system.
Set up a plate document or experiment with the following thermal-cycling conditions:
| A | B | C |
|---|---|---|
| STEP | Temp. (°C) | Time (mm:ss) |
| Hold | 50°C | 02:00 |
| Hold | 95°C | 00:20 |
| PCR (40 cycles) | 95°C | 00:03 |
| 60°C | 00:30 |
Start the run.
Analyze the data
Data analysis varies depending on the real-time PCR system used.
The general process for analyzing gene expression quantitation data involves:
-
Viewing the amplification plots for the entire reaction plate.
-
Setting the baseline and threshold values to determine the threshold cycles (CT) for the amplification curves.
-
Using the relative standard curve method or the comparative CT method to analyze the data.
