cDNA synthesis Tissue

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

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4 years ago

4 years ago by Alex Santos Ribeiro Brazil

cDNA conversion with low concentrations

I have extracted RNA from brain tissue but my RNA concentrations are as low as 5ng/ml with my highest being around 80ng/ml. Will I be able to perform cDNA conversion with concentrations as low as these?

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Found 12 matching solutions for this experiment

Protocol tips
Denature the template-primer mixture by heating the tube for 10 min at +65°C.

Incubate the reaction for 10 to 30 min at +45°C to +55°C.

Inactivate Transcriptor High Fidelity Reverse Transcriptase by heating to +85°C for 5 min
Downstream tips
For downstream PCR, use 1 to 5 μl of the reaction product.

The cDNA can be used for amplification without further purification or manipulation (e.g. RNase H-treatment)
Upstream tips
This kit is optimized for RT reactions using a wide range of total RNA amounts (10 pg -2 μg), such that long and low
abundance cDNAs can be detected by amplification after cDNA synthesis.
Protocol tips
Incubate samples at 45 °C for 30 min.

Incubate samples at 45 °C for 30 min
Downstream tips
The use of higher incubation temperatures up to 48 °C may increase the yield of cDNA synthesized in cases of complex RNA secondary structure
Protocol tips
Heat Oligo mixture to 65°C for 5 min and quick chill on ice and later add reaction components.

Mix and incubate at 42°C for 2 min.

Add 1 μL (200 units) of SuperScript™ II RT and mix by pipetting gently up and down.

Incubate at 42°C for 90 min.

Inactivate the reaction by heating at 85°C for 5 min.
Upstream tips
This Kit is optimized for the
production of targets < 1kb in length.

There will be no loss in functional performance after 20 cycles of freezing on dry ice and thawing on ice
Downstream tips
For downstream reactions, use 1/5th to 1/10th of the first-strand reaction (2-4 μL) for PCR amplification
Upstream tips
This kit can be used to reverse transcribe RNA templates
starting with either total RNA, poly(A)+ mRNA or synthetic transcript RNA
Protocol tips
Anneal reaction at 25°C, and incubate for 5 minutes.

Extend at 42°C for up to one hour

Inactive at 70°C for 15 minutes
Protocol tips
For Genomic DNA elimination, incubate reactions for 2 min at 42°C, then place immediately on ice.

Add Reverse-transcription reaction components and incubate for 15 min at 42°C.

Reverse-transcription reaction components
Protocol tips
Mix RNA sample and primer d(T)23VN and denature RNA for 5 minutes at 70°C.

Add M-MuLV Reaction Mixand
M-MuLV Enzyme Mix and incubate reaction at 42°C for one hour.

Inactivate the enzyme at 80°C for 5 minutes
Protocol tips
After DNAse treatment add Oligo dT Primer,dNTP Mixture,Template RNA and RNase-free dH O and heat at 65°C for 5 min.

Add reaction mix to the above mix and incubate at 30°C 10 min*
and 42C for 60 min
First-Strand cDNA Synthesis Kit 1

GE Healthcare Life Sciences

Protocol tips
Heat the RNA solution to 65ºC for 10 minutes, then chill on ice.

Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour
iScript cDNA Synthesis Kit

Bio-Rad Laboratories

Protocol tips
Reverse transcribe for 20 min at 46C
Downstream tips
For downstream PCR use only 1/10 of the reaction volume (typically 2ul)
Protocol tips
When required, cDNA product can be diluted with 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA
Protocol tips
Follow manufacturer’s instructions
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