RNA Methods of Analyzing Gene Expression

Question 1

Reverse Transcriptase

Answer 1

enzyme from retroviruses that allows the conversion of RNA sequence into DNA, needs a primer and RNA template

Question 2

cDNA generation

Answer 2

double stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by the reverse transcriptase

Question 3

qPCR Principle

Answer 3

cDNA as starting material (reverse transcriptase reaction is included)
reporter emits fluorescence if dsDNA is produced
fluorescence measured with each PCR sample, can be used to quantify amount of DNA used as a template
plateau when reagents get used up
threshold of fluorescence, which one gets there first determines what’s more highly expressed than something that reaches it later

Question 4

qPCR CT value

Answer 4

threshold count: the number of PCR cycles that it takes for the fluorescence to reach the threshold

Question 5

qPCR Cycle #

Answer 5

how many PCR cycles happen

Question 6

qPCR Fluorescent Reporter

Answer 6

something that binds to the dsDNA and emits fluorescence, indicating how much is produced

Question 7

qPCR Use of Standard Curve

Answer 7

made using samples with known input amounts

compare sample to standard curve

Question 8

qPCR Microarray Principle

Answer 8

isolate mRNA from two different cells
convert mRNAs to cDNAs using reverse transcriptase and fluorescently labeled dNTPs
add cDNAs to microarray
fluorescent cDNAs anneal to complementary sequences on microarray/chip
each fluorescent spot represent a gene expressed in the cells, can be in both or one or the other depending on the color