Expert
Reverse transcription
- Reverse transcriptase Convert RNA to
complementary DNA (cDNA)
- RNA-dependent DNA polymerase
- M-MLV from the Moloney murine leukemia virus
AMV from the avian myeloblastosis virus
- Uses RNA as a primer
- Reverse transcriptase has RNAse activity
- RNAse H activity (by nature)
o Degradation of RNA in RNA:DNA hybrid
o Unwanted for synthesis of long cDNA
o Breaking down RNA molecule
- RNAse activity decreased due to mutations
o Use modified reverse transcriptase in the lab
so it doe not break down the RNA template
Reverse transcription – primers
- mRNA has an poly A tail on the 3’end
- OligodT
o Primer that consist of only T
o Binds to the poly A tail
o Disadvantage: When RNA is degraded the
poly A tail is the first to degrade so the
OligodT can only be used when the RNA is
good
o If youre gene is long not everything is
transcribed, when you do a PCR it can be
that your specific primers bind to the ‘5 and
then nothing can be transcribed
o OligodT are very specific
Only reverse transcribe mRNA
- Random hexamers
o Random sequence of 6 oligomers
o Binds tot he hole gene
o More background (rRNA etc)
o Starts at all positions, also near 5’ end
- Gene specific primers
o Bind to your mRNA
o Only quantify one gene per sample
, Reverse transcription samples
- Normalize RNA input
o 300ng – 1000ng
- Choose oligodT or random hexamers or combination
- Controls
o RT- control
Contamination with gDNA no RT
No reverse transcriptase
o no template negative control (NTC)
Contamination of mixture no
RNA
- In total 8 samples for RT
o 6 RNA samples
o 2 controls
o 500-1000 ng of sample is loaded on the
agarose gel
o Put 1 µg for the cDNA syntheses
Genomic DNA contamination
- rDNAse treatment during RNA isolation
- Genomic DNA contains exons and intron and mRNA only contains exons
- Primers during amplification
o Need a primer that is part of exon 1 and exon 2 to detect only cDNA
E1 and E2 are not close in the genomic DNA so the primers can not bind
to this
o Primes that bind only to Exon 1 or 2
Genomic DNA is large so is not transcribed the hole thing
- qPCR DNA polymerase get a small amount of time
PCR
- Dilute cDNA 10-100x! ~10-50 ng of ‘cDNA’ as input
o Rule of thump: 1ug of RNA = 1ug of cDNA
Nanodrop cannot because in the cDNA master mix there are more pasts
of DNA (Primers enzymes)
o Otherwise the DNA polymerase does not work
- Check annealing temperature
- Reference gene (GAPDH) & gene of interest
- Controls?
o gDNA (no RT)
o No RNA
o PCR master mix (no cDNA template control NTC)
- DNA polymerase needs it own environment
Reverse transcription
- Reverse transcriptase Convert RNA to
complementary DNA (cDNA)
- RNA-dependent DNA polymerase
- M-MLV from the Moloney murine leukemia virus
AMV from the avian myeloblastosis virus
- Uses RNA as a primer
- Reverse transcriptase has RNAse activity
- RNAse H activity (by nature)
o Degradation of RNA in RNA:DNA hybrid
o Unwanted for synthesis of long cDNA
o Breaking down RNA molecule
- RNAse activity decreased due to mutations
o Use modified reverse transcriptase in the lab
so it doe not break down the RNA template
Reverse transcription – primers
- mRNA has an poly A tail on the 3’end
- OligodT
o Primer that consist of only T
o Binds to the poly A tail
o Disadvantage: When RNA is degraded the
poly A tail is the first to degrade so the
OligodT can only be used when the RNA is
good
o If youre gene is long not everything is
transcribed, when you do a PCR it can be
that your specific primers bind to the ‘5 and
then nothing can be transcribed
o OligodT are very specific
Only reverse transcribe mRNA
- Random hexamers
o Random sequence of 6 oligomers
o Binds tot he hole gene
o More background (rRNA etc)
o Starts at all positions, also near 5’ end
- Gene specific primers
o Bind to your mRNA
o Only quantify one gene per sample
, Reverse transcription samples
- Normalize RNA input
o 300ng – 1000ng
- Choose oligodT or random hexamers or combination
- Controls
o RT- control
Contamination with gDNA no RT
No reverse transcriptase
o no template negative control (NTC)
Contamination of mixture no
RNA
- In total 8 samples for RT
o 6 RNA samples
o 2 controls
o 500-1000 ng of sample is loaded on the
agarose gel
o Put 1 µg for the cDNA syntheses
Genomic DNA contamination
- rDNAse treatment during RNA isolation
- Genomic DNA contains exons and intron and mRNA only contains exons
- Primers during amplification
o Need a primer that is part of exon 1 and exon 2 to detect only cDNA
E1 and E2 are not close in the genomic DNA so the primers can not bind
to this
o Primes that bind only to Exon 1 or 2
Genomic DNA is large so is not transcribed the hole thing
- qPCR DNA polymerase get a small amount of time
PCR
- Dilute cDNA 10-100x! ~10-50 ng of ‘cDNA’ as input
o Rule of thump: 1ug of RNA = 1ug of cDNA
Nanodrop cannot because in the cDNA master mix there are more pasts
of DNA (Primers enzymes)
o Otherwise the DNA polymerase does not work
- Check annealing temperature
- Reference gene (GAPDH) & gene of interest
- Controls?
o gDNA (no RT)
o No RNA
o PCR master mix (no cDNA template control NTC)
- DNA polymerase needs it own environment
