Few basic questions about RNA - (Mar/29/2011 )
Hello :-)
1. How long RNA can survive at -20 deg, and how many freeze-thaw cycles? If I add Ribolock inhibitors and if I always thaw them on ice, can they survive.....3-4 thawings in the frame of 2-3 days - this is the time I need for all my procedures and combinations.
2. When remove RNA from freezer, how long can we keep it on fresh ice?
2. What is RNA normalization?
3. How someone could estimate the amount of mRNA if it not visible on the gel?
4. If I see the two rRNA bands this would mean that my RNA is intact. Apart from this, what more information gives me the RNA electrophoresis, if I see only the rRNA?
5. If I load my RNA sample on a 1% agarose and I don`t see anything on the level of the upper marker, would it mean that the sample is not being contaminated with gDNA?
6. As far as I undestand, non-PCR-ed gDNA makes something like.....bands with approximate size at about.....8000-10 000 bp, is that correct?
7. Is it necessary to heat RNA/loading buffer mixture before loading on gel?
Answer of any of these questions will be highly appreciated.
Nephrite
You should store your RNA at -80C. Then it will last pretty much indefinitely, as its very stable at that temperature. Even better is to store your RNA in 75% ethanol at -80C. Always keep your RNA on ice when its out on the bench. As long as its on ice and isn't poor quality to begin with, I've had no problem with it out for an hour.
Normalization: Usually if you are looking a specific RNA species you would normalize against another RNA species that is known or proven to be consistently expressed (same level of expression) across all sample types. Normalization accounts for differences in sample amounts. Normalization isn't special to RNA. For example, if I wanted to compare the expression level of RAS between sample 1 and sample 2, I would normalize using actin, since actin is constitutively expressed at the same level. Then this would account for if I had more cells in sample 1 than sample 2 to begin with.
Make sense?
You can also use nanodrop or other spectrophotometer methods to access RNA amount.
Nephrit on Tue Mar 29 10:23:40 2011 said:
Hello :-)
1. How long RNA can survive at -20 deg, and how many freeze-thaw cycles? If I add Ribolock inhibitors and if I always thaw them on ice, can they survive.....3-4 thawings in the frame of 2-3 days - this is the time I need for all my procedures and combinations.
2. When remove RNA from freezer, how long can we keep it on fresh ice?
2. What is RNA normalization?
3. How someone could estimate the amount of mRNA if it not visible on the gel?
4. If I see the two rRNA bands this would mean that my RNA is intact. Apart from this, what more information gives me the RNA electrophoresis, if I see only the rRNA?
5. If I load my RNA sample on a 1% agarose and I don`t see anything on the level of the upper marker, would it mean that the sample is not being contaminated with gDNA?
6. As far as I undestand, non-PCR-ed gDNA makes something like.....bands with approximate size at about.....8000-10 000 bp, is that correct?
7. Is it necessary to heat RNA/loading buffer mixture before loading on gel?
Answer of any of these questions will be highly appreciated.
Nephrite
Thank you very much, bitotechgirl.
So, normalization means, when I compare my cDNA fragments of interest versus the expression of the particular housekeeping gene in all samples? If this is it, I am always doing it.
I thought it is a very special method of RNA quantification on....gel.
Thank you for your useful explanations.
Nephrite
biotechgirl on Tue Mar 29 17:53:23 2011 said:
You should store your RNA at -80C. Then it will last pretty much indefinitely, as its very stable at that temperature. Even better is to store your RNA in 75% ethanol at -80C. Always keep your RNA on ice when its out on the bench. As long as its on ice and isn't poor quality to begin with, I've had no problem with it out for an hour.
Normalization: Usually if you are looking a specific RNA species you would normalize against another RNA species that is known or proven to be consistently expressed (same level of expression) across all sample types. Normalization accounts for differences in sample amounts. Normalization isn't special to RNA. For example, if I wanted to compare the expression level of RAS between sample 1 and sample 2, I would normalize using actin, since actin is constitutively expressed at the same level. Then this would account for if I had more cells in sample 1 than sample 2 to begin with.
Make sense?
You can also use nanodrop or other spectrophotometer methods to access RNA amount.
Nephrit on Tue Mar 29 10:23:40 2011 said:
Hello :-)
1. How long RNA can survive at -20 deg, and how many freeze-thaw cycles? If I add Ribolock inhibitors and if I always thaw them on ice, can they survive.....3-4 thawings in the frame of 2-3 days - this is the time I need for all my procedures and combinations.
2. When remove RNA from freezer, how long can we keep it on fresh ice?
2. What is RNA normalization?
3. How someone could estimate the amount of mRNA if it not visible on the gel?
4. If I see the two rRNA bands this would mean that my RNA is intact. Apart from this, what more information gives me the RNA electrophoresis, if I see only the rRNA?
5. If I load my RNA sample on a 1% agarose and I don`t see anything on the level of the upper marker, would it mean that the sample is not being contaminated with gDNA?
6. As far as I undestand, non-PCR-ed gDNA makes something like.....bands with approximate size at about.....8000-10 000 bp, is that correct?
7. Is it necessary to heat RNA/loading buffer mixture before loading on gel?
Answer of any of these questions will be highly appreciated.
Nephrite
RNA quantification on gel is just an approximation, you can see concentration (very basicaly), you can see integrity of RNA (the bigger 28S rRNA band should be twice as bright as the lower 18S rRNA, if you got more 18S then your RNA is starting to degrade) see this Ambion Tech note, and you can spot gDNA contamination on the top of the gel or still in wells. But to quantify it for accurate qPCR analysis you should use spectrophotometric or fluorimetric methods, another Ambion Tech note.
RNA loading buffer (in our case) contains formamide so the RNA gets denatured chemically and no heating is required, I don't know what protocol for RNA check are you using.