CMMB 549 Lecture Notes - Lecture 25: Rna-Seq, Nanopore, Reverse Transcriptase
28 Dec 2019
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Proteomics: everything involved in translation and protein activity. Tell relative levels of transcript in a cell. Difference: in microarrays, everything is semi-quantitative (ratios), but in rna seq, you"re able to purify individual rna molecule to create a cdna library, then from sequencing you count individual molecules present. Expression based on digital data, its quantitative to determine rna molecules in a cell. Don"t use nanopore for this because cant get large number of reads like in illumina. Use reverse transcription of some variety to get cdna library. Once you do this, you can sequence this dna like any other dna. Isolate rna through molecular biology protocol, random priming using hexamers which allow first strand synthesis to get initiated, then get second strand synthesis to generate cdna library. Rt-pcr: computer program to design primers and amplify sequences. This might go wrongly if you lose strandedness, but this can be controlled.
