biotech siRNA part 2

Question 1

RT-PCR makes how many products?

Answer 1

1

Question 2

RT-PCR allows one to measure the amount of transcript from a single species relative to a control. What would this control be?

Answer 2

Housekeeping gene

Question 3

What is a housekeeping gene?

Answer 3

Doesn’t typically change expression levels.

Question 4

80% of the total RNA is?

Answer 4

rRNA

Question 5

If we were to run out total RNA what would we get on a gel?

Answer 5

Smear of RNA and two bands (28s and 18s) rRNA.

Question 6

What does a weird 28s/18s ratio tell us?

Answer 6

Contamination occured.

Question 7

What strand binds to the mRNA, sense or antisense?

Answer 7

Antisense (sense gets degraded)

Question 8

What sequence do we use for the siRNA control?

Answer 8

scramble sequence

Question 9

Method we used to isolate RNA was what?

Answer 9

GITC based method.

Question 10

What does a chaotrope do?

Answer 10

Denature RNAse.

Question 11

How do we get rid of gDNA contamination?

Answer 11

QIA shredder column.

Question 12

What primer did we use for the RT-PCR?

Answer 12

oligo-dt

Question 13

What primers do we design for the pcr quantitation?

Answer 13

for the TALIN gene, make primers specific to end of one exon and start of another so that gDNA is not amplified.

Question 14

What primers do we make to prevent amplifying gDNA for both GAPDH and TALIN?

Answer 14

primers across intron-exon boundaries.

Question 15

What part of the graph for increased mRNA do we look at to compare expression levels?

Answer 15

linear part

Question 16

When using the software and drawing a box around the bands, the amount of pixels is proportional to what?

Answer 16

amount of product