RT-PCR and quantitative analysis

RT-PCR and quantitative analysis

RNA Extraction: 

Material:

RNAeasy isolation kit (Qiagen, Cat # 74104)

1. Collect cells and wash them once with 1XPBS.

2. Re-suspend cells in 350 µl of RLT buffer.

Note: Proceed or store at -80°C.

3. Process samples according to the Qiagen RNeasy kit protocol.

4. For the elution step, add 15 µl of RNase-free water directly to the spin column membrane. Centrifuge columns for 1min at ≥8000 x g to elute the RNA. The extraction usually yield 50-500ng total RNA for 500-5000 Hu-MuSCs.

Note: Proceed or store RNA samples at -80°C.

cDNA synthesis:

Material:

High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, Cat# 4368814)

1. Prepare 2X RT master mix on ice according to the High-Capacity cDNA Reverse Transcription kit protocol:

2. Prepare a no reverse transcriptase sample to assess DNA contamination.

3. Add 10 µl of 2X RT master mix to 100-200ng of RNA sample.

4. Mix the reactions by briefly vortexing, then centrifuge.

5. Load samples to the thermocycler, use the following program (as indicated by the High-Capacity cDNA Reverse Transcription kit protocol):

6. Dilute cDNA 1:5 (i.e. add 80 µl of RNase-free water per sample).

Note: Diluted reaction products can either be assessed immediately or stored at -20°C for later use.

cDNA pre-amplification

Note: Since we have limited samples due to the low amounts of cell collected we pre-amplified our cDNA. 

Material:

GE PreAmp Master Mix (Fluidigm Inc, Cat# 100-5876 C2)

Samples were processed according to the Fluidigm Preamp Master Mix and Taqman protocol below.

1. Pool the Taqman Gene Expression Assays, as desired. Here we pre-amplified our cDNA for human PAX7, CAV1, GAPDH, b-ACTIN.

Note: The dilution reagent volume should be adjusted based on the amount of Taqman Gene Expression Assay used. The whole volume reaction should be adjusted proportionally based on the number of samples to be amplified.

2. Prepare the sample Pre-Mix and samples as shown below:

3. In a PCR plate, aliquot 3.75 µl of Pre-Mix for each sample.

4. Add 1.25 µl of cDNA to each well containing the Pre-Mix, making a total volume of 5 µl.

5. Mix the reactions by briefly vortexing, then centrifuge.

6. Place the plate in the thermocycler and run the following program:

Note: Cycles can be decrease to 10 cycles or increased to 20 cycles. Run control samples (pre-amp and no pre-amp) in the latter qPCR to verify that the ∆Ct is correctly maintained upon pre-amplification.

7. After cycling, dilute the reaction 1:10 by adding 45 µl of RNase-free water.

Note: Diluted reaction products can either be assessed immediately or stored at -20°C for later use.

Diluted reaction products should be stable for at least one week.

Real-time quantitative PCR

Material:

Taqman Gene Expression Assays (20X)

Taqman universal Master Mix II, with UNG (Cat # 4440038)

Optical 384-well plate (Cat # 4360954)

MicroAmp Optical Adhesive Film (Cat # 4360954)

Note: Each reaction is run in 5 µl and in triplicate

1. In a 96 well plate: 

Add cDNA 3.5X mix to each well

Add Taqman gene assay / Taqman universal Master Mix II to each well

2. Aliquot using a multichannel pipette into the 384-qPCR plate in triplicate.

3. Add MicroAmp Optical Adhesive Film to the qPCR plate.

4. Briefly centrifuge and place the plate in a ViiA 7 thermocycler (Life Technologies):

5. Calculate the average Ct (cycle threshold) using triplicate's Ct value.

6. Calculate the ∆Ct using Beta actin for normalization as endogenous control.

7. Adjust the 2^∆∆Ct so that the relative expression is against the desired sample.