I retro-transcribed total RNA from a fungi with oligoT primers, but I need to check it's a double strand and not a single strand. How could I do it?
Hi Michela....
I have few queries and few suggestion for your problem...
1) Have you done and ss-cDNA synthesis or ds-cDNA synthesis...because ...If reverse transcription is done using oligodT primer....only single strand will be synthesized in the first place ...ds-cDNA willl be done only when you use some other enzymes (DNA pol, RNAseH and DNA ligaes) after completion of ss-cDNA....(there are obviously some exceptions)..So iy you have not done ds-CDNA synthesis....it should be single stranded only..
2) If you have done ds-CDNA synthesis but you are not sure whether you have succeded or not...you can check this by different methods
a) Load both the ss-cDNA and ds-cDNA reactions on agarose gle and you can see the difference....the smear of pool of cDNA is slightly shifted up in the ds-cDNA lane......and you will see good amount of product (smear) in the ds-cDNA lane as it is more efficient that synthesis of first cDNA strand
b) You can check using ssDNA and dsCDNA specific stain (like SYBR I and II) to stain the gels and see the difference
c) Do a hyperchromic shift analysis on spectrophotometer....ssDNA should show more hyperchromic
shift....but it may be difficult to analyze this in a pool of cDNAs....
Hi, in the literature of TTV I found a method, ssPCR, which is strand-specific PCR. It is used to search for the replicative dsDNA form of TTV. If you use this, you get PCR product for both strands in case you have dsDNA but only one of your PCR will have a yield if it is ssDNA. I used it with success. Hopefully I'll find the protocol and can send it later if you think.
Best,
Ferenc
I found it at the end; we adapted the method described in the paper: http://www.ncbi.nlm.nih.gov/pubmed/11793398 to our needs and worked very well. I do not know your system, I only hope, I could help.
Ferenc
Hi Michela....
I have few queries and few suggestion for your problem...
1) Have you done and ss-cDNA synthesis or ds-cDNA synthesis...because ...If reverse transcription is done using oligodT primer....only single strand will be synthesized in the first place ...ds-cDNA willl be done only when you use some other enzymes (DNA pol, RNAseH and DNA ligaes) after completion of ss-cDNA....(there are obviously some exceptions)..So iy you have not done ds-CDNA synthesis....it should be single stranded only..
2) If you have done ds-CDNA synthesis but you are not sure whether you have succeded or not...you can check this by different methods
a) Load both the ss-cDNA and ds-cDNA reactions on agarose gle and you can see the difference....the smear of pool of cDNA is slightly shifted up in the ds-cDNA lane......and you will see good amount of product (smear) in the ds-cDNA lane as it is more efficient that synthesis of first cDNA strand
b) You can check using ssDNA and dsCDNA specific stain (like SYBR I and II) to stain the gels and see the difference
c) Do a hyperchromic shift analysis on spectrophotometer....ssDNA should show more hyperchromic
shift....but it may be difficult to analyze this in a pool of cDNAs....
In Agarose you see only smear, you cannot visualize it because neither you amplify any cDNA nor too high single RNA in high copy number to form bands, you only make a single copy from single RNA. in SYBR 1 or 2 is failure due to common 2ry Structures of RNA.
if you done 2nd strand synthesis steps then only you have to check for ds cDNA ,difficult to analyze this in a pool of cDNAs, and last what are you going to prove with ds cDNA, Go ahead further to your goal.
You all perfectly got my problem! I have a two step process to get firstly sscDNA and then dsCDNA, but I have to use all sscDNA to obtain ds (it's in the kit instruction), so I don't have sscDNA to compare ds anymore...any suggestion?Have you ever try to denature a cDNA?do you think that there's a difference in melting ss and ds and that I colud see this difference on total cDNA?
Thank you so much for your advises!!!!!
Hi Michela....
Although the kits for dscDNA synthesis always say to use complete sscDNA product for the dscDDNA synthesis....but for the purpose of comparison you can always reduce the volumes for the second step....so you store half of the sscDNA and use other half for the dscDNA synthesis and then compare them.....
Alternatively ...if you don't want to play around the volumes...make two tubes for sscDNA and subsequently use one of them for the dscDNA synthesis..
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