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Flashcards in biotech siRNA part 2 Deck (16)
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1

RT-PCR makes how many products?

1

2

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

Housekeeping gene

3

What is a housekeeping gene?

Doesn't typically change expression levels.

4

80% of the total RNA is?

rRNA

5

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

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

6

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

Contamination occured.

7

What strand binds to the mRNA, sense or antisense?

Antisense (sense gets degraded)

8

What sequence do we use for the siRNA control?

scramble sequence

9

Method we used to isolate RNA was what?

GITC based method.

10

What does a chaotrope do?

Denature RNAse.

11

How do we get rid of gDNA contamination?

QIA shredder column.

12

What primer did we use for the RT-PCR?

oligo-dt

13

What primers do we design for the pcr quantitation?

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

14

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

primers across intron-exon boundaries.

15

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

linear part

16

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

amount of product

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