RPKM calculation

[huangjun]

Hi there,
Recently, i began to learn RNA-seq analysis, to some plans, like maize, if we have the reference genome, could anyone tell me how to calculate the RPKM values? I'm thinking that ,first, map the reads to the assembled transcripts; second, from the sam format document, it would give us the transcripts length and the number of mapped reads, i'm not sure my thinking is wright. could anyone tell me the pipline that calculate the RPKM values? Thanks!

[gringer]

First google result for RPKM gives a reasonable formula:

http://www.clcbio.com/manual/genomics/Definition_RPKM.html

[manasarai2009]

Hi
I would like to know how does RPKM helps in knowing absolute number of RNA molecules. I mean for example
Example: 1kb transcript with 2000 alignments in a sample of 10 million reads (out of which 8 million reads can be mapped) will have RPKM 10'9*2000/8*10'6*1000=250

So from this example does it means that 250 RNA molecules of a particular transcript X is present.

Please help me out by explaning in a simple way

[dariober]

This paper Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples makes a good case why transcripts per million (TPM) should be preferred to RPKM.

Also I think TPM gets closer to what manasa is asking.

Dario