Application of RNase - (May/30/2012 )
Hello.
My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.
A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.
I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.
Do I really have to perform this extraction step (which I don`t know how in this small volume)?
Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?
Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.
Thank you.
-Nephrite-
Nephrite on Wed May 30 15:34:26 2012 said:
Hello.
My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.
A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.
I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.
Do I really have to perform this extraction step (which I don`t know how in this small volume)?
Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?
Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.
Thank you.
Even I faced the same problem whenever I used kit I was getting around 100-400ng/ul concentration of RNA and my RNA was degrading because of RNase from some sources. Now I started using Normal manual method and I made solutions in DEPC treated water. Now there is no problem.
I will suggest you go for the phenol choloroform extraction, because this is the best way to get rid off any possible RNase contamination if you have in your RNA and it will concentrate your RNA. With the manual method Now I am getting 1-2ug/uL concentration of RNA.
-ashu2007-
ashu2007 on Wed May 30 15:48:58 2012 said:
Nephrite on Wed May 30 15:34:26 2012 said:
Hello.
My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.
A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.
I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.
Do I really have to perform this extraction step (which I don`t know how in this small volume)?
Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?
Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.
Thank you.
Even I faced the same problem whenever I used kit I was getting around 100-400ng/ul concentration of RNA and my RNA was degrading because of RNase from some sources. Now I started using Normal manual method and I made solutions in DEPC treated water. Now there is no problem.
I will suggest you go for the phenol choloroform extraction, because this is the best way to get rid off any possible RNase contamination if you have in your RNA and it will concentrate your RNA. With the manual method Now I am getting 1-2ug/uL concentration of RNA.
Thank you for your kind and quick reply but I actually want to destroy all RNA in my RNA samples and to measure the gDNA incontaminants :-) The meaning I guess is that it will increase the concentration of gDNA if the RNA has been destroyed. I just wonder do I have to deactivate the enzyme after that :-)
-Nephrite-
Nephrite on Wed May 30 19:55:21 2012 said:
ashu2007 on Wed May 30 15:48:58 2012 said:
Nephrite on Wed May 30 15:34:26 2012 said:
Hello.
My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.
A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.
I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.
Do I really have to perform this extraction step (which I don`t know how in this small volume)?
Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?
Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.
Thank you.
Even I faced the same problem whenever I used kit I was getting around 100-400ng/ul concentration of RNA and my RNA was degrading because of RNase from some sources. Now I started using Normal manual method and I made solutions in DEPC treated water. Now there is no problem.
I will suggest you go for the phenol choloroform extraction, because this is the best way to get rid off any possible RNase contamination if you have in your RNA and it will concentrate your RNA. With the manual method Now I am getting 1-2ug/uL concentration of RNA.
Thank you for your kind and quick reply but I actually want to destroy all RNA in my RNA samples and to measure the gDNA incontaminants :-) The meaning I guess is that it will increase the concentration of gDNA if the RNA has been destroyed. I just wonder do I have to deactivate the enzyme after that :-)
I think RNA can be destroyed by RNAse with in 10 minutes and after that keep it for 10 minutes at 65.C so that whole RNase will be deactivated.
-ashu2007-