Lecture RNA-extraction-RT-qPCR 2024

Real Time
Reverse Transcription quantitative PCR

(RT-qPCR)
Overview:
• RNA in general
• RNA isolation techniques
• RNA quality and integrity
• PCR + Reverse Transcription + RT-qPCR

From DNA to protein:
central dogma of modern biology (F. Crick)
lit.genius.com
Reverse transcription
Total cellular RNA:
>80% ribosomal RNA (rRNA): structural component of ribosomes

10-15% transfer RNA (tRNA): translates mRNA info into the appropriate
amino acid
1-5% messenger RNA (mRNA): template for protein synthesis, reflects gene
expression at a given time
<1% small noncoding RNAs (miRNAs…..)
Why would we study mRNAs?

• Abundance: measure expression levels
• Sequence: predict the protein product
• Size: examine differential splicing
• Dynamics: temporal, developmental, tissue specificity
Cell lysis - breakdown
DNA
RNA
Proteins
Lipids
metabolites
…….
SKMel30 melanoma cells

RNA isolation
Cells
Tissue
Cell lysis
RNA isolation
(affinity purification,
organic extraction)
Check RNA quality

(purity and integrity)
Downstream applications
(RT-qPCR, sequencing …)
Ribonucleases (RNases):
• Naturally occuring enzymes that degrade RNA

• Common laboratory contaminant (bacterial and human sources)
• Also released from cellular compartments during RNA isolation from
biological samples
Protection:
 Wear gloves at all times and change them regularly
 Use RNase-free tubes and pipet tips
 Use dedicated RNase-free chemicals
 Supplement reactions with RNase inhibitors
 Include chaotropic agents (guanidine) in the procedure (by destabilizing
H-bonds, van der Waals forces and hydrophobic interactions, chaotropic agents
destabilize, inactivate and precipitate proteins including nucleases)
Organic extraction of total RNA
Phenol-chloroform extraction (liquid-liquid extraction):
• Adding an equal volume of phenol-chloroform to an aqueous
solution of lysed cells, mixing the 2 phases and allowing the phases
to separate by centrifugation
• Lysis buffer contains detergents and guanidinium isothiocyanate to inactivate

RNases
• Acidic phenol is used because it retains RNA in the aqueous phase, but moves
DNA into the interphase and the phenol phase
• RNA cleaned by alcohol precipitation or column purification
Affinity purification of RNA
Silica/glass-based membranes/filters: selectively bind DNA and RNA, depending

on the salt conditions and other factors influenced by the extraction method
Affinity purification of total RNA
Buffer contains chaotropic

salts and detergents
Binding conditions have

to be adjusted (alcohol)
to remove residual salts
and proteins
Elution buffer (water)

Adapted from Zymo Research Corp.
releases RNA from the silica
RNA quantity and purity measured by spectrophotometry:
260 nm: nucleic acids (DNA and RNA)
280 nm: proteins
230 nm: guanidine, organic contaminants (like phenol)
Quantity: 1 OD260 unit = 40 μg/ml of ssRNA
→ A260 x dilution x 40 = [RNA] μg/ml
Purity: A260/A280: ratio ~2.0 pure RNA
ratio < 1.8 protein contamination
A260/A230: ratio 2.0-2.2 pure RNA
ratio < 1.8 guanidine or organic solvant contamination
RNA integrity based on evaluation of 28S and 18S rRNA
• Agilent Bioanalyzer (combination of microfluidics, capillary electrophoresis
and fluorescent dye that binds to nucleic acids)
• RNA gels
PCR: polymerase chain reaction
~95°C 55-65°C ~72°C
e.g.
230 = 109
Wikipedia
Detection of PCR products
endpoint detection
Detection of PCR products
Real-Time PCR: data collection during the exponential phase

→ signal proportional to number of amplicons generated
Reverse transcription
• Reverse transcriptase (RNA-

dependent DNA polymerase)
• Retroviruses
• Viruses, bacteria, animals and plants:
 Propagation of retroviruses
 Retrotransposition
 Telomeric extension
 msDNA biosynthesis
Thermofisher
Reverse transcription PCR
RNA is converted to complementary DNA (cDNA) by reverse

transcription, and then the cDNA is amplified by PCR
Step 1
1-step 2-step
RT-PCR RT-PCR
Step 2
Microbiologynotes.org
Reverse transcription primers
Only used in
1-step RT-PCR
Oligo(dT) primers:
• Specific for mRNA
• cannot be used for RNAs without polyA tail
• effective with full-length RNA transcription Use a mixture of oligo(dT)
• some 3’ bias can occur and random primers:
Random primers: strength of both without
• can be used with most RNA species their pitfalls
• Some very long RNAs cannot be
completely transcribed
-> truncated cDNA transcripts
Real time quantitative PCR
(qPCR)
• Method for the detection and quantification of nucleic acids (DNA, RNA, miRNAs)
• Advantage over traditional PCR where too much amplification makes it impossible to
quantitate the amount of starting nucleic acid material:
->product accumulation is monitored as the PCR amplification proceeds, at the end of each
cycle, eliminating the problem of endpoint analysis
• Monitoring of the PCR reaction during early & exponential phases where the first significant
increase in the amount of PCR product correlates to the initial amount of target template
RT-qPCR is based on the measurement and quantification of
fluorescence emitted by a reporter molecule in real time
1. Non-specific DNA binding dyes like SYBR green
• SYBR green binds to double-stranded (ds) DNA, but not single-stranded (ss) DNA.
• There is little fluorescence emitted from SYBR green in solution, but it emits fluorescence
very brightly upon binding to dsDNA.
• SYBR green signal intensities correlate with DNA amplified, thus the initial sample
input amounts.
2. Specific probes (Taqman probes)
Reporter
The fluorescence of the reporter dye

is suppressed by the quencher
Probe cleavage by Taq polymerase to free the

reporter dye -> the fluorescence intensity
correlates with initial sample input amounts
(Taq polymerase has 5’―› 3’ exonuclease activity)
Fluorescence
Cycle
Quantification cycle (Cq): metric used for analyzing qPCR results
- represents the number of cycles needed to reach a set threshold fluorescence signal level
- relative value: relative to the starting template copy number
- specific for the instrument and reagents used, the efficiency of the PCR amplification,
the efficiency of cleavage or hybridization of the fluorogenic probe, the sensitivity of
detection.
A lower Cq correlates with a higher amount of starting template and a higher

Cq value correlates with a lower amount of starting template
Endogenous control/Reference genes
• RNA or DNA that is naturally present in each experimental sample

• widely expressed and assumed to be 'constantly expressed‘
• By using an invariant endogenous control as an active 'reference', quantitation
of mRNA can be normalized for differences in the amount of total RNA added to
each reaction and correct for sample-to-sample variations in reverse transcriptase
PCR efficiency, for variation in RNA integrity and cDNA sample loading .
• recommended to use between 2-5 validated stably expressed reference
genes for normalization
Melt curves
For SYBR Green-based amplicon detection, SYBR Green will detect

any double stranded DNA including primer dimers, contaminating DNA,
and PCR products from mis-annealed primers.
With probe-based assays, amplicon specific primers gain an additional
level of specificity from the probe, which is a third sequence that has to
bind within the amplicon of the target sequence in order to generate a
fluorescent signal
Melt curve analysis when

using intercalating dyes
More information about melt
curves, melt curve analysis
and qPCR primer design will
be given in the lecture on
Tuesday
PCR inhibitors
• all substances that have a negative effect

on the PCR reaction
• can originate from the sample or may be
introduced during sample processing or
nucleic acid extraction
• major consequence of a partly or total
Sidstedt et al, 2020

inhibition of the PCR is decreased sensitivity
or false-negative results
• Melanin: known PCR inhibitor, by binding to thermostable DNA polymerase

• Pigment produced by melanocytes protecting cells against DNA damage by
UV light
• melanin present in melanoma tumor cells is often co-purified during DNA
or RNA extraction
Effect of MEKi on melanogenesis
melanocyte
https://doi.org/10.3390/biom9110645
• α-MSH produced by keratinocytes

• CREB: TF
• MITF (melanocyte-inducing TF)
• Tyrosinase (involved in pigmentation), as well as Shah et al, 2017, Scientific Reports
tyrosinase-related protein (gene name TYRP1)

Biological and technical replicates –
What is the difference?
• Biological replicate: RNA/DNA samples extracted from the same cells or tissues treated
always in the same way but in an independent manner; parallel measurements of
biologically distinct samples. A biological replicate is used to verify reproducibility of an
assay. It is used to rule out factors such as culturing conditions, temperature, CO2 conc. etc
as possible variables affecting the assay.
For this reason, biological replicates are performed ONE AT A TIME!
• Technical replicate: RNA/DNA samples extracted from the same source at the same
moment. Differences in measurements are due to technical differences in assay processing.
A technical replicate is only used to monitor variability within 1 experiment.
A good technical replicate is run 3x for each sample!
Technical replicates Biological replicates
Any questions??

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Real Time

Reverse Transcription quantitative PCR

• PCR + Reverse Transcription + RT-qPCR

>80% ribosomal RNA (rRNA): structural component of ribosomes

Why would we study mRNAs?

SKMel30 melanoma cells

Check RNA quality

• Naturally occuring enzymes that degrade RNA

• Lysis buffer contains detergents and guanidinium isothiocyanate to inactivate

Silica/glass-based membranes/filters: selectively bind DNA and RNA, depending

Buffer contains chaotropic

Binding conditions have

Elution buffer (water)

~95°C 55-65°C ~72°C

Real-Time PCR: data collection during the exponential phase

• Reverse transcriptase (RNA-

RNA is converted to complementary DNA (cDNA) by reverse

1. Non-specific DNA binding dyes like SYBR green

The fluorescence of the reporter dye

Probe cleavage by Taq polymerase to free the

A lower Cq correlates with a higher amount of starting template and a higher

• RNA or DNA that is naturally present in each experimental sample

For SYBR Green-based amplicon detection, SYBR Green will detect

Melt curve analysis when

• all substances that have a negative effect

Sidstedt et al, 2020

• Melanin: known PCR inhibitor, by binding to thermostable DNA polymerase

• α-MSH produced by keratinocytes

tyrosinase-related protein (gene name TYRP1)

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