Why is cDNA more stable than mRNA?
cDNA is not subject to RNase degradation, making it more stable than RNA. In RT-PCR, the starting RNA is subsequently degraded, dsDNA is produced, and PCR amplification proceeds in the usual manner.
How much cDNA do you get from 1ug RNA?
If you put 1 ug/ul of RNA in, you’ll get 1 ug/ul of cDNA.
How is RNA converted to cDNA?
The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology.
How much RNA add to cDNA synthesis?
For RNA to cDNA synthesis, we use 1 ug of RNA, then the final (20 ul) reaction mix is added to 80 ul of diH2O (final cDNA concentration 1:5), and our data is beautfiful for qPCR / rt-PCR. You can use 2 ug of total RNA for cDNA synthesis which is good enough for further qPCR studies.
Is cDNA unstable?
cDNA is stable as it’s already DNA.
Is cDNA stable at 4 degree?
The cDNA (prior to cDNA amplification) can be safely stored overnight at 4˚C or -20˚C.
Can you Nanodrop cDNA?
Nanodrop gives fake result of quantification of cDNA . So its better to take Total RNA concentration from nanodrop .
How much cDNA is needed for qPCR?
For optimal performance of the assays, use 1 to 100 ng cDNA per 20 µL reaction.
Why do we need to reverse transcribe the mRNA to cDNA?
Second, PCR amplification only works on DNA, so unless you can obtain enough RNA to feed directly into your sequencing protocol, you need to amplify with PCR, and therefore you must reverse-transcribe to cDNA.
Why is oligo dT an effective primer for reverse transcriptase?
Oligo(dT) primers amplify only mRNAs containing a poly(A) tail, since that is where the primer binds to promote reverse transcription. Random primers amplify most RNA species, including degraded RNA and viral genomes.
How do you measure cDNA quality?
You can check quality of cDNA using either regualr PCR or qPCR. Any housekeeping gene can be used to check the quality of your cDNA, provided primers with different product length of within the same gene can be used in regualr PCR and then run an agarose gel. In qPCR lower the Ct value better the cDNA quality is.