GENET 390: Topic 2.4 cDNA/ Q-PCR/

*****What is cDNA and How is cDNA generated?

DNA synthesized in the lab by RT-PCR or Reverse transcriptase PCR

• complementary copies of the RNA sequences

Do cDNA contain Introns? Why or Why not?

only the exons

• represents only the genes that were actively expressed (transcribed into mRNA) in the cell at the time the RNA was collected.

****Why cDNA?

1. MORE stable than mRNA

• Once reverse-transcribed into cDNA, the sequence is preserved in a stable DNA form for cloning or sequencing.

2. is the Spliced variant specifically expressed in that tissue or condition or timing

What is a cDNA Library?

Collection of clones produced by RT-PCR to be stably maintained in E.coli

• Now all in SILICO

What Enzyme is used to generate cDNA + what is it isolated from?

Reverse Transcriptase

• isolated from Retroviruses

******What are the 3 main differences between cDNA + gDNA libraries

1. cDNA doesn’t have introns = ONLY EXPESSED sequences. 

• gDNA = Entire genome

2. cDNA = SPECIFIC to Tissue At specific time (Differ between tissues + developmental time)

• gDNA = Identical in all tissues

3. Differ in PRIMERS

*****How do cDNA + gDNA differ in the primers used.

cDNA = uses POLY T primers

gDNA = uses random primers

****What are POLY T Primers?

oligo-dT primer (a string of T’s) that binds to that poly-A tail.

• This ensures only mRNA is copied (not rRNA or tRNA, which lack poly-A tails).

*****What is a limitation of cDNA not having Introns?

No REGULATORY SEQUENCES

What feature of cDNA makes it possible to study splice variants of a gene?

cDNA is made from mature mRNA, so its sequence directly shows which exons were joined during splicing.

• Can look at alt. splicing

What are the steps for cDNA synthesis? (eukaryotes)

1. Isolate total RNA from cell (all RNA not just mRNA)

2. Attach Poly T primer

3. Polymerize the DNA using mRNA as a template (first strand synthesis)

4. Terminal tailing

5. Attach Poly T primer to first strand of RT synthesized DNA

6. Polymerize (2nd strand synthesis)

7. PCR amplification of double strand DNA product

****How does Amplification to cDNA differ depending on if you have isolated PROKARYOTES vs. EUKARYOTES?

Prokaryotes = NO POLY A tail

• Make fake poly A tail

• or use random primers

Eukaryotes = YES poly A tail

• Use POLY T primer

*****What adds the Fake Poly A tail to prokaryotes + to the first copy of DNA for Eukaryotes?

TdT

****What synthesizes the new cDNA?

Reverse transcriptase

***What acts as both the FORWARDS + REVERSE primers for eukaryotes?

OligoT-Primer/ Poly T primer

What is qPCR? what is its purpose?

quantitative Polymerase Chain Reaction (also called real-time PCR).

• amplifies DNA and also monitors the amplification using fluorescent signals.

Purpose = how much DNA was present initially

• Measure the level of transcription of a gene by measuring how many copies of DNA were initially present

PCR vs. RT-PCR vs. qPCR vs. qRT-PCR

PCR = PCR

RT PCR = Reverse transcription (cDNA) PCR

qPCR = Quantitative/real time pcr

qRT-PCT = q pcr + RT pcr Combined

****To determine the relative abundance of transcript relative to control you need to compare MANY TRANSCRIPTS at once. What is a normal amount of tests + replicates to run?

96 or 384 well plates

do experiment in TRIPLICATE

****Fluor are used to QUANTIFY PCR product at the end of each PCR cycle. What are the 2 most common?

1. SYBR green

2. TaqMan

****What does SYBR Green bind to?

dsDNA

• no fluorescence when bound to ssDNA

****What is the Theory behind how SYBR green works to Quantify initial DNA?

SYBR green fluorescence intensity should directly correlate to amount to PCR present

WHY should SYBR green fluorescence intensity should directly correlate to amount to PCR present

because it binds to dsDNA at a CONSTANT RATIO

***There are 2 ISSUES with using SYBR green. What are they?

1. Slowly inhibits PCR rxn

2. Binds DNA but also bind primer dimers

****how does SYBR green slowly inhibit PCR rxn?

Due to its physical INTEGRATION into DNA, it gets in the way of the polymerization

*****How to deal with SYBR green binding to PRIMER DIMERS?

Preform a melt curve as a control for primer dimer formation

****How do you go about performing a melt curve?

At the end of the cycles, the cycler gradually heats up the sample from annealing temp to 94 degrees

• Read fluor signals @ each temp to see when each ds DNA melts into ss DNA when fluorescence drops

****What results do you WANT and what do you NOT WANT?

Want = SINGLE PEAK around 80 degrees (desired product melting temp)

NOT want = 2 PEAKS indicates primer dimers

Why are 2 peaks a bad result. What does it signify about the fluorescence detections

Not useful results bc all fluorescence detection are an overestimate

• contamination with other DNA

***What is used for the POSTIVE control of qPCR?

Housekeeping gene

• confirms amplification worked

****What are Housekeeping genes + how do they let you confirm amplification worked?

Genes that are Always present (needed for basic cell functions)

• They are expressed at relatively constant levels across most cell types and conditions

Allows to quantify absolute/known number of transcripts

What are some examples of common positive control?

Histones, Actin, GAPDH, B-Tubulin, 18s rRNA

****What are NEGATIVE CONTROLS for in qPCR?

Control for contamination RNA or DNA

****What are some examples of possible Negative controls?

• No RT (NRT)

• No template

• No Taq

***What is NRT

No reverse transcriptase = should not see any amplification of product

*****What is a very common contaminant in RT-PCR?

gDNA

• When you extract total RNA from cells or tissues, traces of DNA from the genome often remain.

• If not fully removed (e.g., by DNase treatment), that DNA can be amplified during PCR.

Issue bc desired = RNA

****What is a way to test for gDNA contamination?

Design primers so that they span a INTRON/EXTRON boundary

• Amplified gDNA will be longer than expected from RT product. due to the presence of INTRONS

• cDNA does not contain introns due to splicing of mRNA

*****What principle does TaqMan use to quantify?

FRET (fluorescence resonance energy transfer)

****How does FRET work?

If two fluor’s are close together (one quencher + one fluor) there is NO signal

If the dyes are further apart that the quench is release then a signal is detected

****How does FRET work with qPCR?

As DNA polymerase moves along template, 5’-3’ exonuclease, breaks the interactions between quencher + reporter (on ssDNA probe)

= Separate quencher from reported = fluorescence 

• Amount of fluorescence = approximate of # of times probe has bound + then degraded

****What are the ssDNA probes?

sequence of DNA that ANNEALS TO TARGET SEQ + contains a both a fluor + a quencher

***is an issue with ssDNA probe that it competes with primer for extension?

NO

• Probe = NON EXTENDABLE = not competing

****Why is the DNA Probe NON-EXTENDIBLE?

No 3’ OH

******How are qPCR results measured for both SYBR green + TaqMan?

Threshold cycle/ Ct

*****What is Ct?

Cycle at which the amplification signal exceeds the background

• # of cycles of PCR required for a detectable fluorescence (aka. above background)

****How does Ct change as transcript abundance in OG sample increases?

More Initial RNA template = reach threshold faster

= less cycles

****We always run qPCR multiple times, WHY might we get slightly different Ct numbers each time?

Amount of starting template = crucial + is ERROR PRONE

***What are some potential errors that may cause the variation in amount of starting template affecting Ct in replications of qPCR?

Vertexing

Pipetting

Elution volume etc. etc.

*****What are the 2 options we have with dealing with the slightly variable qPCR data?

1. Absolute quantification

2. Relative quantification

****How to perform ABSOLUTE QUANTIFICATION? How does it work?

Compare measured with Control (housekeeping genes)

• Obtain Ct values of control + Plot against log copy number

• Produce STANDARD CURVE of control using a 10x dilution

• Compare measured Ct data with the standard curve

eg. if measured Ct = 25 than there were 1000 copies transcript of OG (figure B, blue line)

****How to perform RELATIVE QUANTIFICATION? How does it work?

Relative expression of desired transcript by comparing it to two controls

• Read notes for more info