Now I need to determine the subcellular localization of a given lncRNA with qPCR after cellular fractionation.However,I am not sure whether same amount(e.g.1ug) or same proportion(e.g.1%) of nuclear and cytoplasmic RNA should be used for reverse transcription,and how to caculate the cytoplasmic to nuclear ratio with Ct values from respective cDNAs.Thanks.
http://bitesizebio.com/19087/how-rna-fish-can-complement-qpcr-and-vice-versa/
If the goal is to "determine the subcellular localization of a given lncRNA ", I would perhaps try to incorporate RNA FISH into your experimental design. My impression from your question, is that you will get very limited information with qCPR as your readout for localization. A northern Blot could help answer if differing species of the given lncRNA exist in nucleus vs cytoplasm.
Ratios:
https://www.ncbi.nlm.nih.gov/pubmed/16893657
Hello
i agree with Soufiane Aboulhouda .
See link . Good luck
http://www.sciencedirect.com/science/article/pii/S1874939915002163
Although I do agree that RNA FISH will give you the real picture of an RNA sub-cellular localization, I also think that qPCR could give an indication. Particularly in case of strongly nuclear enriched lncRNAs. What you normally do is to compare the expression of your lncRNA with the expression of an RNA for a coding gene. In that case, because you measure a relative intracellular localization, you don't have to care about the % of RNA is in your fraction relative to the total. I have used equal amounts of RNA for cDNA synthesis, so that you are sure that cDNA efficiency will be the same. After qPCR you can calculate the relative amount of your transcript using the formula: 2^Cp-refence gene/2^Cp-lncRNA (assuming that primer efficiency for both reference gene and lncRNA is 2). You have to keep in mind that this will be an indication only and that your result will be different if you use a different reference gene, because also mRNA for coding genes can have different localizations. I have used Malat or Neat as nuclear markers and HPRT as cytosolic marker. This approach has been used in several publication. But, again, RNA FISH (if it is working) is giving you the best answer.
Hope this help, good luck,
Ingram
@Ingram Laccarino, agreed! All depends on what and how much information you would like to get out of the experiment, and how much time you are willing to invest. As indicated, FISH is not as trivial as qPCR
It does not matter if you use equal amounts of cytoplasmic and nuclear RNA. What is important is to use a marker gene for both the cytoplasmic RNA (e.g. GAPDH) and the nuclear RNA (e.g., MALAT-1) and normalize the expression of your gene of interest relative to both markers. Make sure you also use the expression level from total RNAs as a point of reference.
Good luck
BA
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