January 2, 2016

Dr. Campbell and Team's Experimental Protocol

• Subject 6 Burmese Python's to a starvation period.
• Feed 3 Pythons and kill each 30 minutes after food was no longer visible in the mouth.
• Also, kill the 3 remaining fasted snakes.
• Harvest, weigh, and flash freeze organs immediately after death. Flash-freezing prevents organ degradation.
• Use kit to transcribed cDNA from 100 micrograms of RNA sampled from 0.1 grams of small intestine and liver tissue from each snake.
• Fragment RNA and combine with RNA primer, identification code, and hexamer to produce sufficient cDNA library.
• Amplify DNA samples; cut out samples at 500bp; reamplify
• Use FastQC data analysis to determine quality of data.
• Use DEseq software package to normalize expression for all reads (normalize for length of gene per million reads).

Questions to consider:

• Sampling problem:
  • Did our sample come from the right part of the organ?
  • How do we know we did not sample connective tissue?

• What do you gain by fractioning the mRNA into short fragments randomly?
• How do you prime every mRNA individually?

Preliminary Research

Snake Small Intestine Gene Search

• Organic Cation Transporters in Intestine, Kidney, Liver, and Brain
• The Function of Gp170 the Multidrug Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa (Abstract)
• Conditional (intestinal-specific) Knockout of the Riboflavin Transporter-3 (RFVT-3) Impairs Riboflavin Absorption
• Also consider the KEGG pathway.

Ashlyn's Main Page

Scrap Notes

• Figure out what ScreenShots are at the bottom of the page.
• Elaborate on FPKM?
• Make sure you got everything from your first notes.

-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