Hi,
How RT-qRCR can help me to IDENTIFY a lncRNAs? if i have deferentially expressed profiles...
Dear Nono, if I understand you well, You ask about an real time PCR approach for long non protein coding RNAs identification? If yes, it is quite simple. Sorry if I am not correct in my conclusion...
1. Design specific primers and probes (TaqMan) to enlarge specifity.
2. Extract total RNA.
3. Do quality control at least in agarose gel.
4. Measure concentration (with florescence if you can)
5. Do reverse transcription with random oligos (hexamrs, decamers, etc.).
6. Do Real Time PCR optimization for each primer+probe set.
7. Prepare 5-fold serial dilutions of your cDNA (5-6 steps) to receive callibrators for standard curve building and effectiveness estimation for absolute quantitation. If you want to use E^-ddCq method you should choose at least a pair of stable expressed referense genes under your experimental conditions. It is step number 0, prior of step 1 in my list.
8. Do real time with your samples and analize data.
Because of npcRNAs does not have exon/intronic borders you should use DNAase during your purification or phenol extraction (not so many DNA molucules after purification).
I hope it would help you.
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