January 2, 2016 - Revision history

Asgruber at 19:27, 12 February 2016

2016-02-12T19:27:33Z

Asgruber: /* Dr. Campbell and Team's Experimental Protocol: */

2016-02-12T19:27:15Z

‎Dr. Campbell and Team's Experimental Protocol:

Asgruber at 18:16, 12 February 2016

2016-02-12T18:16:12Z

Asgruber: /* Snake Small Intestine Gene Search */

2016-02-12T18:13:08Z

‎Snake Small Intestine Gene Search

Asgruber: /* Snake Small Intestine Gene Search */

2016-02-12T18:12:04Z

‎Snake Small Intestine Gene Search

Asgruber: /* Snake Small Intestine Gene Search */

2016-02-12T18:11:12Z

‎Snake Small Intestine Gene Search

Asgruber at 18:03, 12 February 2016

2016-02-12T18:03:35Z

Asgruber at 17:56, 12 February 2016

2016-02-12T17:56:35Z

Asgruber at 16:37, 12 February 2016

2016-02-12T16:37:27Z

Asgruber: Created page with "== 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 fro..."

2016-02-12T16:20:13Z

Created page with "== 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 fro..."

New page

== 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

@@ Line 20: / Line 20: @@
 *What do you gain by fractioning the mRNA into short fragments randomly?
 *How do you prime every mRNA individually?
 = Preliminary Research =

@@ Line 1: / Line 1: @@
-== Dr. Campbell and Team's Experimental Protocol: ==
+== Dr. Campbell and Team's Experimental Protocol ==
 *Subject 6 Burmese Python's to a starvation period.
@@ Line 20: / Line 20: @@
 *What do you gain by fractioning the mRNA into short fragments randomly?
 *How do you prime every mRNA individually?
 = Preliminary Research =

@@ Line 28: / Line 28: @@
 *[https://www.researchgate.net/publication/21808741_The_function_of_GP170_the_multidrug_resistance_gene_product_in_the_brush_border_of_rat_intestinal_mucosa The Function of Gp170 the Multidrug Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa] (Abstract)
 *[http://ajpgi.physiology.org/content/ajpgi/early/2015/12/04/ajpgi.00340.2015.full.pdf Conditional (intestinal-specific) Knockout of the Riboflavin Transporter-3 (RFVT-3) Impairs Riboflavin Absorption]
 *Also consider the KEGG pathway.

@@ Line 26: / Line 26: @@
 === Snake Small Intestine Gene Search ===
 *[http://www.annualreviews.org/doi/full/10.1146/annurev.physiol.60.1.243?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed Organic Cation Transporters in Intestine, Kidney, Liver, and Brain]
-*https://www.researchgate.net/publication/21808741_The_function_of_GP170_the_multidrug_resistance_gene_product_in_the_brush_border_of_rat_intestinal_mucosa The Function of Gp170 the Multidrug Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa] (Abstract)
+*[https://www.researchgate.net/publication/21808741_The_function_of_GP170_the_multidrug_resistance_gene_product_in_the_brush_border_of_rat_intestinal_mucosa The Function of Gp170 the Multidrug Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa] (Abstract)
 *[http://ajpgi.physiology.org/content/ajpgi/early/2015/12/04/ajpgi.00340.2015.full.pdf Conditional (intestinal-specific) Knockout of the Riboflavin Transporter-3 (RFVT-3) Impairs Riboflavin Absorption]
 *Also consider the KEGG pathway.

@@ Line 26: / Line 26: @@
 === Snake Small Intestine Gene Search ===
 *[http://www.annualreviews.org/doi/full/10.1146/annurev.physiol.60.1.243?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed Organic Cation Transporters in Intestine, Kidney, Liver, and Brain]
-*https://www.researchgate.net/publication/21808741_The_function_of_GP170_the_multidrug_resistance_gene_product_in_the_brush_border_of_rat_intestinal_mucosa The Function of Gp170 the Multidrug-Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa] (Abstract)
+*https://www.researchgate.net/publication/21808741_The_function_of_GP170_the_multidrug_resistance_gene_product_in_the_brush_border_of_rat_intestinal_mucosa The Function of Gp170 the Multidrug Resistance Gene Product in the Brush Border of Rat Intestinal Mucosa] (Abstract)
 *[http://ajpgi.physiology.org/content/ajpgi/early/2015/12/04/ajpgi.00340.2015.full.pdf Conditional (intestinal-specific) Knockout of the Riboflavin Transporter-3 (RFVT-3) Impairs Riboflavin Absorption]
 *Also consider the KEGG pathway.

@@ Line 25: / Line 25: @@
 === Snake Small Intestine Gene Search ===
-*[[Organic Cation Transporters in Intestine Kidney Liver and Brain]]
+*[http://www.annualreviews.org/doi/full/10.1146/annurev.physiol.60.1.243?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed 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]]

@@ Line 10: / Line 10: @@
 *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: ===
@@ Line 21: / Line 22: @@
 = Preliminary Research =

← Older revision		Revision as of 16:37, 12 February 2016
Line 1:		Line 1:
−	== Dr. Campbell and ~~team~~'s Experimental Protocol: ==	+	== 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 =
		+

	-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.		-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.