cDNA synthesis Cell lines

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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Found 12 matching solutions for this experiment

Upstream tips
The
enzyme maintains activity at 42-50 °C and is suitable for
synthesis of cDNA up to 13 kb.
Protocol tips
Use following conditions: 25°C for 5 min, 55°C for 30 min and 75°C for 10 min
Downstream tips
The reverse transcription reaction product can be directly
used in PCR applications or stored at -20 °C for less than
one week. For longer storage, -70 °C is recommended
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 50 min.

Inactivate the reaction by heating at 70°C for 15 min.
Protocol tips
Prepare transfection master mix and mix throughly by pippetting.

Add 15ul master mix to 5ul of RNA for each RT reaction.

Adjust the volume of water if the input of RNA is not to 5 ul input (1ug-1pg).

Transcribe for 30 min at 42 °C and the reaction is inactivated for 5 min at 85 °C.
Protocol tips
Prepare the reverse-transcription master mix on ice and later incubate for 60 min at 37°C.

Incubate for 5 min at 95°C to inactivate Quantiscript Reverse Transcriptase and later use it for downstream experiments
Upstream tips
This Kit uses the random primer scheme for initiating cDNA synthesis.
Protocol tips
Add 10 μL of 2X RT master mix and 10 μL of RNA sample and mix.

Treat tubes at 25C for 10 minutes, 37C for 2h and 85C for 5 minutes.
Protocol tips
Heat mixture to 65°C for 5 minutes and quick chill on ice and incubate at 37°C for 2 minutes.

Incubate 50 minutes at 37°C.

Inactivate the reaction by heating at 70°C for 15 minutes

SuperScript IV VILO Master Mix

Thermo Fisher Scientific

Upstream tips
Use up to 2.5 μg of total RNA as starting material in a 20-μL reaction
Protocol tips
Treat the mixture at 25 °C for 10 min, incubation at 50 °C for 10 min, and incubation at 85 °C for 5 min.
Upstream tips
THis kit can be used to synthesize first strand cDNA from total or polyA RNA.

Highly sensitive: use less of your precious RNA samples
Protocol tips
Heat at 65°C for 5 min, then immediately cool on ice
Upstream tips
Use 1 to 10 ng of total RNA per 15- µL RT reaction
Protocol tips
Treat RT mixture for 30 minutes at 16C , 30 minutes at 42C and to inactivate treat the tube at 85C for 5 minutes
Protocol tips
Follow manufacturer's instructions
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