Although it’s called RNA seq, very rarely (but becoming more common) is RNA itself actually being sequenced. Instead, cDNA is. mRNAs or total RNA is reverse transcribed to get cDNA (more on this below) which is ligated (stitched to) end adapters that allow it to be sequenced. This will tell you “all” that’s present - but if there are only a few things you’re interested in, it’s much simpler to just search for their cDNAs directly using RT-qPCR, where you make lots of copies of it (if it’s present) and use fluorescence to measure the copies as they’re made. The more you start with, the faster the signal will rise so it will tell you about how many copies there were. Here’s some more detail on this, adapted from a much longer post of it… blog form: bit.ly/rtrtqpcrprimer; RUclips: ruclips.net/video/kp4ZX2lOr6w/видео.html the first step in RT-qPCR (after you isolate the RNA) is making DNA copies of the RNA copies of the DNA recipes through REVERSE TRANSCRIPTION. Normal transcription goes DNA->RNA. REVERSE transcription goes RNA->DNA. It uses a different polymerase (instead of the usual DNA-RNA or DNA-DNA Pols you need an RNA-DNA Pol - we call such Pols reverse transcriptase) - and we call the DNA copies of the mature mRNAs complementary DNA (cDNA)
The reverse transcriptase can make DNA copies of RNA, but it still has the limitation of needing a double-stranded starting platform - so you need to provide primers for it.
Usually you want to measure multiple mRNAs. Even if you’re only interested in levels of one, you need to normalize it to something so you make sure that if you see twice as many of jt under a set of conditions it isn’t just because you had RNA from twice as many cells.
Traditionally this is done by comparing levels of “housekeeping genes” which are recipes that are made at pretty constant levels under all conditions
Since you want to count multiple things, you usually start by stabilizing and reverse-transcribing all the mRNA and/or all the RNA (mRNA or otherwise). To just RT the mRNA you can take advantage of that generic poly-A tail we saw earlier. Since A pairs with T, you can use a short stretch (usually 15) of DNA T’s (an oligo(dT)) as an all-mRNA-specific primer. It’ll latch onto the poly-A tail to provide a starting point for the reverse transcriptase.
If you provide a “normal” oligo-dT primer, it can latch on anywhere along the poly-A tail, but if you use an “anchored oligo-dT” which ends (3’ end) with a letter other than T (a G, C, or A that acts as an anchor) - it can only latch onto the part closest to the end of the unique stuff (binds at the 5’ end of the poly(A) tail.
To illustrate: imagine you have an mRNA that’s unique part ends in a C
blahblahblahCAAAAAAAAAAAAAAAAAAAA
If you use and un-anchored oligo(dT) like TTTTTTTT that can bind anywhere along the stretch of As and serve as a primer for the reverse transcriptase. So you can get
Although it’s called RNA seq, very rarely (but becoming more common) is RNA itself actually being sequenced. Instead, cDNA is. mRNAs or total RNA is reverse transcribed to get cDNA (more on this below) which is ligated (stitched to) end adapters that allow it to be sequenced. This will tell you “all” that’s present - but if there are only a few things you’re interested in, it’s much simpler to just search for their cDNAs directly using RT-qPCR, where you make lots of copies of it (if it’s present) and use fluorescence to measure the copies as they’re made. The more you start with, the faster the signal will rise so it will tell you about how many copies there were.
Here’s some more detail on this, adapted from a much longer post of it… blog form: bit.ly/rtrtqpcrprimer; RUclips: ruclips.net/video/kp4ZX2lOr6w/видео.html
the first step in RT-qPCR (after you isolate the RNA) is making DNA copies of the RNA copies of the DNA recipes through REVERSE TRANSCRIPTION. Normal transcription goes DNA->RNA. REVERSE transcription goes RNA->DNA. It uses a different polymerase (instead of the usual DNA-RNA or DNA-DNA Pols you need an RNA-DNA Pol - we call such Pols reverse transcriptase) - and we call the DNA copies of the mature mRNAs complementary DNA (cDNA)
The reverse transcriptase can make DNA copies of RNA, but it still has the limitation of needing a double-stranded starting platform - so you need to provide primers for it.
Usually you want to measure multiple mRNAs. Even if you’re only interested in levels of one, you need to normalize it to something so you make sure that if you see twice as many of jt under a set of conditions it isn’t just because you had RNA from twice as many cells.
Traditionally this is done by comparing levels of “housekeeping genes” which are recipes that are made at pretty constant levels under all conditions
Since you want to count multiple things, you usually start by stabilizing and reverse-transcribing all the mRNA and/or all the RNA (mRNA or otherwise). To just RT the mRNA you can take advantage of that generic poly-A tail we saw earlier. Since A pairs with T, you can use a short stretch (usually 15) of DNA T’s (an oligo(dT)) as an all-mRNA-specific primer. It’ll latch onto the poly-A tail to provide a starting point for the reverse transcriptase.
If you provide a “normal” oligo-dT primer, it can latch on anywhere along the poly-A tail, but if you use an “anchored oligo-dT” which ends (3’ end) with a letter other than T (a G, C, or A that acts as an anchor) - it can only latch onto the part closest to the end of the unique stuff (binds at the 5’ end of the poly(A) tail.
To illustrate: imagine you have an mRNA that’s unique part ends in a C
blahblahblahCAAAAAAAAAAAAAAAAAAAA
If you use and un-anchored oligo(dT) like TTTTTTTT that can bind anywhere along the stretch of As and serve as a primer for the reverse transcriptase. So you can get
can you make cDNA from RNA genome (virus) as template instead from mRNA ?
yes. you can make cDNA from any RNA, we just commonly make it from mRNA