PCR

Question 1

Differences between RNA & DNA

Answer 1

DNA: - double-stranded - deoxyribose vs. ribose sugar - DNA is less reactive because of C-H bonds, RNA is more reactive because of C-OH (hydroxyl) bonds - base pairs: A-T & C-G vs A-U & C-G

Question 2

qPCR on mouse hippocampus steps:

Answer 2

1. dissection - sacrifice mouse - isolate hippocampus - homogenize tissue in TRIzol with bead beater 2. RNA isolation - Phase separation - RNA precipitation - washing of pellet - check RNA content & purity 3. cDNA synthesis - using primers, ATP, TTP, GTP, & CTP - a thermic cycle (at 65C RNA denaturates, at 25C the primer anneals, 37C - cDNA is synthesized, 70C - reaction terminated) 4. qPCR

Question 3

How do we isolate RNA?

Answer 3

RNA isolation: - Phase separation Phenol & chloroform are added to the sample, which then undergoes centrifugation, creating 3 phases - bottom - proteins & lipids in phenol, second - DNA in chloroform, upper - RNA in aqueous phase. - RNA precipitation: Isopropanol is added to the sample. RNA cannot dissolve in isopropanol, and is, therefore, dissolved into a white pellet - Washing of pellet - RNA content & purity Purity is checked with Nanodrop (a spectrophotometer) at three wavelengths: 260nm for RNA & DNA, 280nm for proteins/phenol, and 230nm for TRIzol

Question 4

How is cDNA synthesized?

Answer 4

cDNA synthesis: - using primers and ATP, TTP, GTP, & CTP - a thermic cycle (at 65C RNA denaturates, at 25C the primer anneals, 37C - cDNA is synthesized, 70C - reaction terminated)

Question 5

How much of the RNA extracted is actually mRNA?

Answer 5

• around 5%; the other 95% are rRNA & tRNA

Question 6

What are the phases of qPCR?

Answer 6

• Denaturation; cDNA strands fall apart (90C) - Annealing - primer attaches (ca. 60C) - Elongation - synthesis occurs (ca. 72C)

Question 7

The output curves of qPCR are called…

Answer 7

…amplification curves

Question 8

What types of controls are used with qPCR?

Answer 8

1. Positive control 2. Blank (H2O) (to check that the primers & polymerase/SYBR Green mixes are not contaminated) 3. RT- control (for genomic DNA contamination)

Question 9

What is the name of the gel we used for gel electrophoresis?

Answer 9

• Agarose gel

Question 10

Oligo dT vs random primers:

Answer 10

• Oligo dT primers always attach at the poly-A tail on the 3’ end of the transcript - if the gene is long, they may fall off before completing its transcription - random hexamer primers then to give a higher cDNA yield; may overrepresent the central part of the transcript

Question 11

The thermal cycler detects:

Answer 11

… the fluorescence emitted by the excited fluorochrome, is it is caught between cDNA strands

Question 12

What is the melting point?

Answer 12

• the point at which 50% of the DNA is single-stranded

Question 13

What is a melting curve?

Answer 13

• The melting curve we receive is the negative 1st derivative of the melting sequence: this curve shows the negative slope of the melting line - from completely flat to very steep - If there is a small peak earlier on and the main peak is still in the right place, then the first peak is probably from the primer dimers, which means the data might still be usable. Often primers will make this dimers, and that is to be expected.

Question 14

What is a primer dimer?

Answer 14

A Primer dimer (PD) is a potential by-product in PCR, a common biotechnological method. As its name implies, a PD consists of primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.

Question 15

What is the main difference between genomic DNA and cDNA?

Answer 15

cDNA does not contain the introns, which were spliced out in mRNA

Question 16

What is a housekeeping gene?

Answer 16

• a ‘standard’ gene expressed in large quantities in most cells (e.g. ACTB β-actin (common cytoskeletal enzyme); UBC ubiquitin C; etc.)

Question 17

What is genomic contamination and what does it signify?

Answer 17

Genomic contamination (visible in the RT-) occurs when genomic DNA left in the sample is being amplified. This usually signifies a problem with the design of the primer and can be prevented by designing primers on two exons of a gene, since the intron between them will only be present on genomic DNA and not on cDNA. In this case the band in the gel will come up at a different time (because the DNA molecules are larger, as the primer spans an intron, and more fluorescence gets trapped there).

Question 18

What is indicative of primer dimerization?

Answer 18

Primer dimers are usually smaller than cDNA copies are may be seen as low-molecular-weight bands on the agarose gel.

Question 19

Formula for the concentration of a gene of interest at a specific CT (cycle threshold) value:

Answer 19

Xn = X0 * (1+E)^n

where X - concentration of the gene of interest

n = the CT value

E - efficiency; (100% efficiency is an efficiency of 2 - each cycle the amount doubles)

X0 - concentration at the start (lower than concentration at threshold)

Question 20

Explain how to derive the efficiency of the amplification procedure.

Answer 20

Efficiency depends on the concentrations of X (gene of interest) and R (reference gene) at the start of the procedure, and the critical threshold values. To derive efficiency, we need the slope of the line formed by the Logs of the starting concentrations of X (X0) and the corresponding critical threshold values. This slope is then used in the formula E=10^(-1/slope), giving us a number like 1.80, which signifies an efficiency of 80%.

P.S. X0/R0 = 1 * (1+E)^ctR-ctX

Question 21

What is the difference in terms of primer action between the first few cycles?

Answer 21

• During the first cycle only the forward primer binds and copies (because there is no cDNA strand for the backward primer to bond to). In the second cycle, both primers start to bind.

Question 22

If there is a factor 10 difference between concentrations of cDNA, what is the expected difference in cycles?

Answer 22

Because of the exponential nature of the procedure, one would expect to lower concentration to come up 3-4 cycles later (2*2*2=8; 2*2*2*2 = 16)