I need to synthesize cDNA of different plant species and I have relatively low concentrations of RNA (minimum of 15 ng).
I have never done this procedure.
Different people have been suggesting me the use of different kits:
FirstChoice® RLM-RACE (Ambion 1700)
Retroscript Kit (Ambion)
RevertAid First Strand cDNA Synthesis Kit (Fermentas)
And the last, said to be the best (but not explained to me why it is so)
SMARTer RACE cDNA Amplification Kit (Clontech)
Hi Tania,
Here in our lab, we use SuperScript™ III Reverse Transcriptase (Invitrogen). Usually, 1Ug of RNA is good enough to get cDNA. It's will be better to check the quality and integrity of your RNA before proceeding. This kit is straight forward and don't take much time to do. Good luck.
Hi Tania, I used the SMARTer RACE cDNA Amplification Kit from Clontech. I used RNA from guts of Cydia pomonella larvae (they are really tiny). I had good results with this kit. Good luck. Cheers, Nadine
Hi Tania,
Here in our lab, we use SuperScript™ III Reverse Transcriptase (Invitrogen). Usually, 1Ug of RNA is good enough to get cDNA. It's will be better to check the quality and integrity of your RNA before proceeding. This kit is straight forward and don't take much time to do. Good luck.
Hi Tania,
In our laboratory, we try to examine the mRNA expression of immune genes in aquatic animals. We synthesize cDNAs from total RNA of 2.5 micro grams extracted from animals with PrimeScript™ II 1st strand cDNA Synthesis Kit from TaKaRa following the standard protocol of manufacturer [See: http://www.westburg.eu/en/site/products/genomics-research/pcr-rt-pcr/rt-pcr-kits/first-strand-cdna-synthesis-kits/primescriptii-1st-strand-cdna-synthesis-kit/$54524]. This would just take only 1 hour and 20 min. This approach really works well and I guarantee that you would succeed. But, you suppose to quantify and evaluate the purity of your RNA prepared, prior to start the cDNA synthesis. Good luck.
Hi, in our lab we study gene expression in plants and good RNA and cDNA synthesis is very important. we used to use SuperScript™ but now we are using iScript cDNA synthesis kit from Bio-rad. it is very easy to set up (just a reaction mix and a reverse transcriptase to mix with your RNA), allows up to 15 µl of RNA sample in a 20 µl cDNA synthesis reaction and the total input RNA can vary between 1 µg–1 pg. and also, it is very fast (about 40 mins)...i agree with Abdalla.... always chech quality and integrity of RNA before, ans also treat your RNA samples with a DNAse treatment before the cDNA synthesis to degrade any DNA contamination in your sample of RNA.
- Badges
- Science method