Molecular Methods: RNA Purification

• Molecular methods encompass the basic principles, applications, and health and safety considerations of techniques like DNA/RNA extraction and purification, DNA sequencing, basic DNA cloning, PCR, and RT-PCR.

RNA

• Messenger RNA (mRNA) synthesis dynamically expresses the genome of an organism.
• mRNA is central to the information flow within a cell.
• Isolate RNA to examine differential splicing by analyzing size.
• Sequence RNA to predict protein product.
• Measure expression levels by analyzing abundance.
• Analyze temporal, developmental, and tissue specificity through dynamics of expression.
• Total RNA purification involves isolating RNA from other cellular components.
• Cells or tissues must be disrupted rapidly and efficiently when isolating total RNA.
• RNases need to be inactivated when isolating total RNA.
• Denature nucleic acid-protein complexes when isolating total RNA.
• RNA must be selectively partitioned from DNA and protein when isolating total RNA.
• Different tissues/sources present different issues for total RNA isolation.

RNases

• Wear gloves during RNA extraction at all times.
• Use RNase-free tubes and pipette tips.
• Use dedicated, RNase-free chemicals.
• Pre-treat materials with extended heat(180°C for several hours).
• Wash with diethylpyrocarbonate (DEPC)-treated water, NaOH or H2O2
• Supplement reactions with RNase inhibitors.

Guanidinium Method

• A method of homogenizing tissue.
• Add Trizol to denature proteins, including guanidine thiocyanate, phenol, and chloroform.
• Centrifuge the sample.
• Isolate total RNA from the upper layer after centrifuging.

mRNA Isolation

• Utilizes the fact that mRNA molecules have a polyA tail.
• Oligo(dT) probes purify mRNA from other RNAs.
• mRNA can be eluted from oligo(dT) matrix using water or low-salt buffer or magnets.

RNA Analysis

• Quantitation of mRNA is a key part of RNA analysis
• Northern blotting:
  • Utilizes hybridization with a gene-specific probe to find abundance and size of the RNA molecule in a sample.
• Ribonuclease protection assay is a method of performing RNA Analysis
• RT-PCR:
  • A highly sensitive/simple method of RNA analysis.
  • Can discriminate closely related mRNAs.
  • It can be difficult to get truly quantitative results using conventional PCR.

Polymerases

• RNA polymerase synthesizes RNA using a DNA template.
• Reverse transcriptase synthesizes DNA using an RNA template.
• Thermostable nucleases, like Taq polymerase, are used for PCR.
• Reverse transcriptase is produced by cells infected with retroviruses like HIV.
• Reverse transcriptase makes DNA (cDNA) from an RNA template.
• cDNA can be inserted into a vector to create a cDNA library.

Molecular methods

• It is important to visualise DNA that has been collected
  • Agarose gel electrophoresis
• Cut or join DNA back together
  • Restriction enzyme digestion
• Make more of DNA
  • Polymerase chain reaction (PCR)
  • Cloning
• Read the DNA
  • Sequencing

PCR

• PCR amplifies a piece of DNA between 2 known sequences.
• Amplification occurs exponentially which means the number of copies = (2 to the power of n), here n = number of cycles.
• The amplified product can be seen in an agarose gel.

PCR Reaction Mix

• It is important to add all ingredients at the start of the reaction.
• Include Deoxynucleotide triphosphates like dNTPs, dCTP, dATP, dGTP, and dTTP.
• PCR Primers are synthetic oligonucleotides.
• DNA to be amplified as a template.
• Use a buffer with magnesium ions.
• Taq polymerase is important for reaction.
• Use very pure water

PCR Primer

• A primer serves as the starting point for DNA synthesis.
• Primers are usually short, chemically synthesised oligonucleotides, with a length of about 20 bases.
• Primers hybridise to a target DNA, which the polymerase then copies.
• Minimum primer length used in most applications is 18 nucleotides.
• Replication starts at the 3'-end of the primer, and copies the opposite strand.
• The primer for DNA synthesis and replication is a short strand of RNA in most cases of natural DNA replication.

PCR Procedure

• Cycle 30 times:
  • 1 minute at 94°C - Denatures DNA
  • 1 minute at 40-60°C - Primers Anneal
  • 1 minute at 72°C - Primer extension
• Amplification should be exponential over 30 cycles, giving 230 (1 X 10º) copies

Applications of PCR

• Study patterns of gene expression.
• Assist DNA sequencing.
• Traditional process of DNA cloning.
• Phylogenetic analysis of DNA from ancient sources
• Study patterns of genetic mapping (RFLP)
• Parental testing to match an individual with their close relatives.

MIC PCR

• Faster PCR reactions.
• Heating and cooling a PCR block takes time typically
• Centrifugation based is important.
• Magnetic induction technology heats the samples and then fan forces air to cool.
• 35 cycles in 35 mins vs 1hr 25mins

Beyond PCR? - LAMP

• The loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA for specific amplification.
• A strand-displacing DNA polymerase initiates synthesis.
• Two specially designed primers form "loop" structures to facilitate subsequent rounds of amplification through extension on the loops with additional primer annealing.
• Isothermal – one temp 65C, no cycling, no PCR machine.
• Bst DNA polymerase is use– rather than Taq polymerase.