Difference between revisions of "January 2, 2016"
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Revision as of 16:20, 12 February 2016
Dr. Campbell and team's Experimental Protocol:
-Flash freeze organs after harvest because RNA is unstable and we do not want it to degrade. -100 micro grams of RNA from 0.1g tissue. RNA isolated from just a kit-- think about sampling problem. Did it come from the right part of the organ? How do we know it's not connective tissue? Dr. C's best may not have been good enough... -You don't sequence RNA, you sequence cDNA! mRNA template --> transcribed to more stable version of cDNA. -Beads with oligonucleotide complementary to part of mRNA. Remove beads, now you just have mRNA. Use Reverse Transcriptase to transcribe cDNA. -RNA FRAGMENTATION: You get very short reads (75bbp). What do you gain by fractionating the mRNA into short fragments randomly? --Now we get a lot more reads (more edges to read from, roughly the same size). -BUT how do you prime every mRNA individually? -generate every possible heximer (6bp), attach it to code (A, B, C... for each snake "xxx") and attach both to primer.
Good DNA samples. Amplify. Cut out at 500bp and reamplify. Reamplification is also at 500bp = really good cDNA library. Resources for snake intestine gene search: ORGANIC CATION TRANSPORTERS IN INTESTINE, KIDNEY, LIVER, AND BRAIN [1] P-glycoproteins anion transporter and OATP P-glycoprotein (MDR1 - humans; mdr1a - mice) GENE?
The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa. [2]
useDEseq is the software package we used to normalize expression for all millions of reads (normalize for length of gene per million reads) FPKM column in excel geneResult we read FPKM and normalize data. Smal DNA chunks, take all of that info and turns little genes into a big gene I don't think multi-drug resistant receptors are really applicable to snakes... but could be a good place to start? Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption. [3] RFVT-3 protein (product of the Slc52a3 gene) riboflavin transporter Also look at KEGG pathway File:Screen Shot 2016-02-02 at 2.23.57 PMMedia:Screen Shot 2016-02-02 at 2.23.57 PM
