Lie Chen and Michael J. Shipston
10. Apply supernatant from step 9 to a QIAprep spin column and centrifuge at 14,000×g for 1 min at room temperature
3.4. Notes
6. It is essential to completely remove all growth medium; oth-erwise this can reduce RNA isolation and purification at later stages by interfering with efficient RNA binding to RNeasy membrane. An optional rapid wash step with PBS can be used, but again all PBS must be removed to avoid dilution.
7. We prefer to use the direct lysis method rather than removing cells by trypsinisation followed by pelleting. Not only does the latter take longer, and include more steps, but thedetach-ment of cells and rounding up can cause dramatic changes in RNA expression during the time required to pellet cells before lysis.
8. An alternative approach with tissue culture cells is to extract cytoplasmic RNA and remove genomic DNA by pelleting cell nuclei. This can help if genomic DNA contamination is an issue but the procedure results in cells being exposed to conditions under which RNase activity may affect the mRNA profile before the lysate is homogenised in guanidium thiocyanate containing buffer. For this procedure add 175 µl of pre-cooled Qiagen cell lysis buffer (RLN) to lyse the cell membranes. Triturate and repeatedly wash the RLN buffer across the well to ensure the complete removal of cells by holding the plate at a 30° angle to allow the solution to drain to the base of the plate. The solution should clear rap-idly indicating immediate cell lysis. Transfer the lysate to an RNase-free Eppendorf tube pre-cooled on ice/water bath and incubate at 4°C for 5 min to ensure complete lysis of cells. Centrifuge the lysate at 300×g for 2 min at 4°C to pellet the nuclei and large cell debris. Transfer the supernatant to a fresh RNase-free Eppendorf tube and proceed from Sub-heading 3.3.2.2, step 2.
9. Many protocols recommend using a rubber policeman/cell scraper to remove lysed cells. We do not recommend this as highly efficient lysis/cell removal is achieved by triturating cells from wells and significant amounts of material can be lost in small wells by adherence to the scraper.
10. While discarding flow through, it is convenient to place the filter in an RNase-free Eppendorf in a rack for convenience while handling the collection tube. As RW1 and RPE buffer contain guanidium thiocyanate do not dispose of these buffers using bleach or strong acid.
11. This relationship is only valid at neutral pH in a low salt buffer. Typically dilute 4 µl of the final RNA eluate from an RNAeasy column in 996 µl 10-mM Tris–HCl, pH 7.0 to achieve an absorbance reading between 0.1 and 1.0. If the reading is above or below these values, adjust the RNA input to ensure reliability of measurements.
12. The ratio is influenced by pH and should be performed in low salt buffers. Ratio is optimally determined at pH 7.5 in these cases. However use of the same RNA dilution in neutral 10-mM Tris–HCl saves time and RNA and gives a reliable indication of purity.
13. Formaldehyde is toxic and is volatile. Ensure that the gel solution is cooled, but not starting to set, before addition.
14. RNA loading buffer is used to denature the RNA; increase the density of the sample to ensure that it sinks into the well (ensure that you do not have residual ethanol as this will cause the sample to float out of the well and furthermore ensure that wells are well washed) and add dyes that migrate through the gel to monitor the extent of electrophoresis.
RNA gels are run at neutral pH, using MOPS buffer that has high buffering capacity at pH 7.0, as RNA is susceptible to degradation at high pH and RNA has a lower pKa than DNA. However, single stranded RNA can display a high sec-ondary structure, which is why formaldehyde is used to keep the RNA denatured throughout.
15. RNA will tend to smear down the back of the Eppendorf tube and appear colourless. To aid orientation, place the Eppendorf so that the hinge is pointing to the outside of the rotor; the RNA will then smear down the wall of the tube below the hinge.
16. Avoid overdrying or heating to speed evaporation. Over-dried RNA pellets can be difficult to re-suspend in water and elevated temperatures can accelerate RNA degradation.
17. Many protocols include addition of 2.5-mM EDTA final concentration to stop reaction. However, care must be taken while doing this, as too much EDTA can reduce the efficiency of subsequent reverse transcription reactions and heat inacti-vation is sufficient. Do not exceed the time/temperature of inactivation as otherwise the RNA may become degraded.
18. Omniscript reverse transcriptase is an RNA-dependent DNA polymerase (reverse transcriptase) enzyme that drives first strand cDNA synthesis. The enzyme also contains RNase H activity that degrades RNA in duplex with cDNA, thus removing the parental RNA template from the RNA:DNA hybrids. If we are isolating RNA from very small samples where recovered RNA may be <100 ng then we routinely use Sensiscript reverse transcriptase from Qiagen.
19. Three different types of primers can be used (1) oligo-dT primer that anneals to the 3′ polyA tail of mRNAs. However, subsequent analysis of splice variants at the 5′ end of the mRNA may be misrepresented due to reduced efficiency for long transcript length (especially if mRNAs for the target
of interest are >1.5 kb); (2) random primers are short (typi-cally hexamers) sequences of DNA that bind along the RNA molecule and thus reverse transcribe polyA+ and polyA− transcripts. In general random primers can produce shorter length cDNAs but with good coverage across the mRNA; (3) gene specific primers can be used if only one target is being amplified; this can improve sensitivity but careful choice of primer is necessary to ensure good representation of subse-quent cDNA and besides other targets cannot be amplified in the subsequent PCR. If using gene specific primers, use a final concentration of 1 µM.
20. DEPC can inhibit the RT reaction. So ensure that if RNase-free water was treated with DEPC, that it has also been well autoclaved to remove any residual DEPC.
21. While Taq DNA polymerases do have a higher error rate as compared to proof reading polymerases such as Pfu, Taq polymerase is required to incorporate overhanging adenines to amplicons to facilitate efficient downstream TOPO clon-ing. It is possible to use a mix of Taq and proof reading enzymes. However, when amplifying amplicons of <1 kb, and analysing multiple amplicons followed by sequencing, we have not experienced any PCR error rate problems by using com-mercial Taq enzymes as compared to hybrid formulations.
22. Buffer and cycling parameters must be optimised prior to use. We typically design primers to have an annealing temperature (Tm) of 60°C and then run optimisation PCRs using 10–50 ng of an appropriate plasmid cDNA template at several different magne-sium concentrations (e.g. final MgCl2 of 5, 3.75, 2.5, 1.25 mM) and annealing temperatures below the Tm of the primers (Tm
−3°C, Tm −5°C, Tm −7°C and Tm −10°C conditions).
23. Use a clean cap-closing tool rather than gloved fingers to signif-icantly reduce chances of cross-contamination between tubes.
24. This is a useful control to ensure that PCR amplification has occurred. However considerable care must be taken as this plasmid stock is a major source of potential contamination.
Inclusion of this control is an excellent way of also testing whether your experimental procedures are effectively pre-venting cross-contamination.
25. Use a thermocycler with a heated lid to avoid use of over-laying mineral oil. This significantly reduces the likelihood of cross-contamination. Whereever possible, arrange tubes with one tube spacing between neighbouring tubes across the PCR block to further reduce contamination crossover.
26. For high efficiency shot-gun subcloning into pCRII TOPO vector, do not freeze samples but use immediately. For very low abundance variants, an additional round of PCR amplification
may be required. In this case, use a 1/100 dilution of reac-tion from step 10 above and use as template in a new PCR amplification from step 2. While the same primers can be used, a set of internally nested primers is preferred.
27. SYBR® Safe is a non toxic alternative to ethidium bromide and very useful for routine DNA amplicon screening. However, if you suspect low abundance amplicons use EtBr to stain the DNA gel using the precautions outlined in Note 1.
28. Ensure that you retain an aliquot of the fresh PCR reaction for downstream ‘shot-gun’ TOPO cloning. Do not mix all the PCR reaction with the DNA loading buffer. When process-ing multiple samples it is convenient to mix the PCR sample with the DNA loading buffer on top of a piece of nescofilm to avoid using additional Eppendorf tubes for mixing. Place multi-ple aliquots of DNA loading buffer in order of sammulti-ple addi-tion to well on nescofilm. Then using a fresh tip each time mix 10 µl of the PCR reaction with the DNA loading buffer on nescofilm by triturating several times and aliquot the mix into the respective well of the DNA agarose gel.
29. It is not necessary to set up a negative control (i.e. no TOPO reaction) for TOPO cloning, because a subsequent PCR screening of colonies will exclude false positive clones. TOPO reactions can be stored at −20°C for long term storage but from our experience, use of the fresh reaction for transforma-tion generates fewer false-positive clones. We find the pCRII TOPO vector very efficient for subcloning amplicons <1 kb;
however, if cloning amplicons larger than 2 kb, we find that the pCR2.1 TOPO vector gives improved cloning efficiency.
30. This approach can be used even when the amplicons are not well separated as the different amplicons are subsequently ligated and characterised in the pCRII TOPO vector. How-ever, where possible, take samples from well isolated bands to improve the efficiency of direct TOPO cloning or re-PCR.
31. Some apparently low abundance high molecular weight DNA amplicons observed on DNA agarose gels may in fact be artefacts. For example, it is possible that heterodimerisation and hairpin looping of DNA fragments may occur, resulting in retarded migration of the DNA fragment. The presence of very large (i.e. several kb) DNA fragments in DNA agar-ose gels of PCR amplicons generally indicates genomic DNA contamination.
32. Pre-warm LB-agar plates (10-cm diameter petri dish containing 50-µg/ml of ampicillin as antibiotic selection) by inverting and placing in a 37°C incubator with the plate slightly over-lapping the lid at one edge. Before use ensure that the con-densate is shaken from the lid and place the plate upright.
33. A convenient spreader can be fashioned from a glass Pas-teur pipette by bending the fine tip in a flame, so that the spreader is approximately half the diameter of the LB-agar plate. Slightly bend the tip upwards to prevent the spreader gouging into the agar. Store the spreader in 100% alcohol and flame between each use, allow to air cool under the aseptic technique (e.g. near a yellow Bunsen flame). It is often convenient to use two plates per transformation; plate one at 50 µl, the other at 200 µl to ensure that discrete well separated colonies are available after overnight incubation.
34. It is important to completely remove all supernatant, as otherwise the efficiency/purity of subsequent plasmid DNA is compromised. Invert the Eppendorf tube on a tissue paper to drain completely. The supernatant should be exposed to bleach before disposal.
35. The rapid neutralisation results in bacterial chromosomal DNA-forming insoluble aggregates while the plasmid DNA covalently closes via interstrand rehybridization and thus remains in solution. The potassium acetate also results in SDS precipitation, which pellets insoluble chromosomal DNA aggregates and protein. Rather than using a phenol/
chloroform extraction, for the sake of convenience we routinely use a Qiagen QIAprep spin miniprep DNA filter to isolate the plasmid DNA from the supernatant, which is produced by precipitation of the insoluble aggregates.
36. Up to 5-ml of bacterial culture can be processed using this method if required, to produce sufficient DNA for downstream applications.
37. Using the alkaline lysis and the DNA miniprep spin column approach outlined in Subheading 3.3.4.5 we find that the miniprep DNA is of high enough quality for automated DNA sequencing using the pCRII TOPO sequencing primers.
Sequence multiple plasmid clones in which different sizes of splice variant amplicons have been determined. Sequence both strands to verify the sequence.
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