MIMM 385 Lecture Notes - Lecture 3: Chaotropic Agent, Rna Extraction, Isothiocyanate
26 Apr 2019
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MIMM385 Lecture 3
RT PCR to assess Gene Expression
3 steps
• RNA extraction
• cDNA synthesis
o Because PCR uses DNA as template
• Real Time PCR
RNA Extraction
Silica-based method
Nucleic acids (both RNA and DNA) bind tightly to silica-based materials (e.g. beads composed of silica
glass) in the presence of chaotropic salts, such as guanidinium isothiocyanate, whereas other less-
charged cellular components do not. Guanidinium isothiocyanate also protects RNA from RNases.
Chaotropic: breakdown/denature structures
Inhibitors of enzymes (RNases) that can disrupt RNA extraction
RNases can be found everywhere, especially on your body and hands
Cells or tissue lysate are passed through a silica-based material in a small column and washed.
The RNA and DNA bind to the silica while proteins and other cellular constituents pass through. (Washed
out)
After washing, the DNA is eluted selectively, although not completely removed, with low salt in the
presence of 35% ethanol. Contaminating DNA is removed by on-column (RNase-free) DNase I treatment
under high salt conditions. Subsequently, after washing away the DNase and salt, RNA is eluted with
water.
RNA Quantification
Nanodrop
C = A/(e * l)
C: the concentration of the nucleic acid in μg/ml
A: Absorbance (OD260)
l: width of the cuvette which used to hold the solution, in cm.
e: Extinction coefficient which is: 40µg/ml for single stranded RNA at OD 260 nm
RNA Purity
The OD260/OD280 value should be in the 1.8 to 2.0 range. If the value is lower: protein contamination.
OD280 - measurement of protein concentration
The OD260/OD230 ratio is helpful in evaluating the carryover of components containing phenol rings
such as the chaotropic salt guanidine isothiocyanate and phenol itself, which are inhibitory to enzymatic
reactions. A260/A230 ≈ 2.0
Value close to 2.0 means that the extraction is of high purity, has low contamination.
This is important as the salts could interfere with the PCR reaction
RNA Integrity
RNA integrity is assessed on an agarose gel (and subsequent staining) or on an instrument such as a
bioanalyzer.
Agarose gel would show bands for 28S and 18S rRNA (cytoplasmic rRNA)
Smear in between the two bands are the mRNA
Not in specific bands as they are of different sizes
Agilent Bioanalyzer: This instrument uses capillary electrophoresis to analyze nucleic acids. It requires
only 25 ng of RNA input. A 2:1 ratio in the area under the peaks for 28S and 18S rRNA indicates intact
total RNA. Degradation is indicated by less pronounced peaks for 28S and 18S rRNA and a 28S:18S rRNA
ratio significantly less than 2:1.
RNA Extraction
*! Watch out for RNases!
• Gloves should always be worn when preparing RNA and should be changed frequently.
• Care should be taken to proceed through the RNA isolation as quickly as possible.
• Work surfaces and micropipettors should be wiped and kept clean.
cDNA Synthesis
The polymerase chain reaction utilizes a DNA polymerase that requires DNA. RNA should therefore be
transcribed into complementary DNA (cDNA).
This is done as PCR uses DNA as template.
cDNA is synthesized using a an enzyme: a reverse transcriptase (process known as reverse transcription)
Components
RNA
Reverse Transcriptase
Primers
dNTPs (C, G, A, T)
Buffer, RNase inhibitors and stabilizers
Important:
Gene expression analysis should reflect the starting amount of RNA transcripts.
The product of cDNA should be proportional to the starting number of RNA transcripts.
Why is it important to maintain a proper proportion?
Over representation of RNA that are abundant, and some RNA are not well transcribed as they are
present in lesser amounts.
Thus, the cDNA that is synthesized should be proportional to the starting amount of RNA transcripts.
To maintain the ratio:
Utilize RNase H+ reverse transcriptase enzyme, which ensure that only one cDNA is synthesized from
one RNA molecule (preserve the 1:1 ratio of RNA to cDNA).
RNA transcript is degraded as the cDNA is synthesized so that further cDNA synthesis from the RNA
transcript is prevented. This is important for RNA transcripts that are not very abundant.
Too much RNase H activity can drastically reduce the yield and ratio of full-length cDNA, which
translates to poor sensitivity.
RNA Priming
3 different types of primers:
Oligo (dT): always initiate reverse transcription at the 3' end of the transcript.
Disadvantage: Difficult secondary structure may lead to incomplete cDNA generation.
Random primers: using hexamers, they anneal throughout the transcripts ensuring a more complete
coverage of the RNA. Higher chance of complete cDNA synthesis.
Disadvantage: Often, hexamers which can lead incomplete cDNA.
Another primer that can be used: Sequence-specific primers
Primer specific for TNF
Disadvantage: The same has to be done with every gene that needs to be amplified and the control
genes. Therefore, it is not very cost-efficient.
Solution: Combination of random primers (longer than hexamers) and oligo(dT) primers -> increases
cDNA synthesis quality.
No-RT Control Reactions
Negative control: to control for DNA contamination in sample.
Controls to prove that signal obtained from experimental samples represent the transcript of interest
(validating specificity).
All real time PCR experiments should include a no-reverse transcriptase control (no-RT control).
No-RT reactions contain all reaction components except the reverse transcriptase.
