Real-time PCR analysis

Request a Protocol: “Real-time PCR analysis”

1.    Collect samples to an RNase free 1,5mL tube containing an appropriate amount of RA1 buffer (according to NucleoSpin RNA KIT)

2.    Isolate the RNA according to manufacturer’s protocol

3.    Elute RNA with 20μl H₂O and measure the concentration and quality via NanoDrop Spectrophotometer (ratio absorbance 260/280nm)

4.    Use 1000ng of RNA per sample for cDNA synthesis (via cDNA reverse transcriptase KIT)

5.    Perform reverse transcription under following conditions

6.    Dilute cDNA 1:5 in DEPC water

7.    Dilute the primers in the following way (inhouse designed, generated from Quiagen)

a.    add an appropriate amount of DEPC water to lyophilized primers to reach a 100uM stock solution

b.    dilute primers further 1:10 in DEPC water

8.    Put 1µl of cDNA for each sample on a clear 384-well plate in triplicates

9.    Add 9µl of Master Mix per well

10. Seal the plate with a cover foil and insert the plate into the Light Cycler 480 machine

11. run RT-PCR under the following conditions

Analysis of raw data

1.    Export the “Crossing Point” (CP-value) of each sample

2.    Generate the average of each CP-value from triplicates

3.    Calculate the relative Quantification via reference gene (GAPDH) of each sample

△CP= 2^- (CP target gene – CP housekeeping gene)

4.    Values can be normalized by setting the control group as 1 and calculating the relative changes

Material