Lie Chen and Michael J. Shipston
3.2. Materials
3.2.1. Isolation of Total RNA from Tissues and Cells
channel. (a) Pore-forming α-subunit of large conductance calcium- and voltage-activated potassium (BK) channels encoded by the KCNMA1 gene. The C2 site of splicing is located in the intracellular C-terminus of the channel. Alternative pre-mRNA splicing in the mouse at site C2 results in five different splice variants at this site as illustrated schematically.
In the ZERO variant exon 19 is spliced to exon 23, in variants e20, e21 (also known as STREX) and e22 the respective exon is included between exons 19 and 23. Open boxes indicate constitutive exons whereas shaded boxes indicate alternatively spliced exons identified in mouse. The number in each box refers to the respective murine exon. Note that exon 23 was previously considered as a constitutive exon; however, analysis revealed a novel variant in which exon 23 is skipped (∆e23). This results in a frame shift and a premature truncation of the protein (see ref. 15). The PCR screening strategy employed a forward (fwd) PCR primer located in exon 15 and a reverse (rev) primer in exon 25 to span several putative exons and introns. (b) Representative SYBR® Safe stained DNA agarose gel (as in Subheading 3.3.3.3) illustrating size fractionation of different sized PCR amplicons. This corresponds to the different site C2 splice variants, resulting from the PCR screen identified using the strategy outlined. (c) Translation of the DNA sequence of identified PCR amplicons reveals the different amino acid sequences (single amino acid code) resulting from the alternative splicing decisions for each variant. Amino acids in bold indicate the respective splice variant specific insert sequences.
1. RNase-free water: add 0.1% DEPC to 18-MΩ quality distilled water in an RNase-free bottle, shake vigorously and leave to stand overnight with loose fitting cap. Autoclave for at least 15 min on a standard wet-cycle. Aliquot small working stocks (~10 ml) for day to day use in RNA extraction procedures.
Discard working stock regularly.
2. Qiagen RNeasy mini spin column kit (includes spin columns, buffers RLT, RW1 and RPE, Cat. no. 74104).
3. Ethanol: use 96–100% ethanol stored in small aliquots at room temperature.
4. Ultrapure agarose: have separate stocks for RNA and DNA gels.
5. 10-mM Tris–HCl, pH 7.0 made up in RNase-free water:
store in small aliquots at room temperature.
6. RNA gel buffer (RGB): prepare 10× stock in RNase-free water with 200-mM 3-[N-morpholino]propanesulfonic acid (MOPS), 50-mM sodium acetate and 10-mM EDTA, pH to 7.0 with NaOH. Autoclave and store at room temperature.
7. RNA gel running buffer (RGRB): prepare 1-L fresh solution containing 100 ml of 10× RNA gel buffer, 20 ml of 37% (12.3 M) formaldehyde and 880 ml of RNase-free water in a fume hood.
8. RNA loading buffer (RLB): prepare 10 ml of a 5× stock solution in RNase-free water containing 4 ml of 10× RNA gel buffer, 20% glycerol, 33% formamide, 4-mM EDTA, pH 8.0, 0.2% bromophenol blue, 0.2% xylene cyanol. Store as 250-µl aliquots at −20°C or at 4°C for up to 3 months.
9. 0.5% SDS: 0.5% (w/v) sodium dodecyl sulfate in RNase-free water.
10. Ethidium bromide (EtBr): prepare 10-mg/ml stock in RNase-free water and store in dark bottle at 4°C. Caution - mutagenic (see Note 1).
11. Sodium acetate: prepare a 3-M stock solution in RNase-free water and adjust pH to 5.2 with acetic acid. Store as 0.5-ml aliquots at −20°C.
12. Cell lysis buffer (RLN, optional): 50-mM Tris–HCl, pH 8.0, 140-mM NaCl, 1.5-mM MgCl2, 0.5% (v/v) Nonidet P-40.
Prepare with RNase-free water.
13. RNase-free DNase 1: store reconstituted enzyme at 2–3 Kunitz units (KU)/µl in RNase-free water and 10× DNase 1 reaction buffer in small aliquots at −20°C.
1. Qiagen Omniscript RT kit (Cat. no. 205111).
2. Random hexamer primers: prepare a 100-µM stock by re-suspending in RNase-free water. Prepare 10-µl aliquots and store at −20°C.
3.2.2. First Strand cDNA Synthesis and PCR Amplification of Splice Variants
3. Oligo-dT primer: prepare a 10-µM stock by re-suspending in RNase-free water. Prepare 10-µl aliquots and store at −20°C.
4. RNA inhibitor (e.g. RNAsin from Promega, Cat. no. N2111):
store at −20°C. Dilute to 10 U/µl with RNase-free water, immediately prior to use.
5. dNTP mix (dATP, dCTP, dGTP, dTTP): prepare 25-mM stock of each dNTP in RNase-free water. Prepare 10-µl aliq-uots and store at −20°C.
6. Taq DNA polymerase (Invitrogen, Cat. no. 18038-042): includes 10× PCR buffer, 50-mM MgCl2 and Taq DNA polymerase.
7. Forward gene specific PCR primer: re-suspend in dH20 and dilute to 10 pmol/µl. Store as 25-µl aliquots at −20°C.
8. Reverse gene specific PCR primer: re-suspend in dH20 and dilute to 10 pmol/µl. Store as 25-µl aliquots at −20°C.
9. Autoclave RNase-free water.
10. DNA gel buffer (TBE) (10×): 0.89-M Tris base, pH 8.3, 0.89-M boric acid, 2-mM EDTA, pH 8.0. Autoclave and store at room temperature.
11. DNA loading buffer (10×): 50-mM Tris–HCl, pH 7.6, 60%
glycerol and 0.25% (w/v) bromophenol blue. Store at room temperature in 1-ml aliquots.
12. SYBR® Safe DNA gel stain (10,000× concentrate, Invitrogen, Cat. no. S33102) store at room temperature in small aliquots.
1. pCRII TOPO, or pCR2.1 TOPO cloning kit (Invitrogen, Cat.
no. K4650-01 or K4500-01): includes TOPO vector and 6×
TOPO Salt buffer (1.2-M NaCl and 0.06-M MgCl2) with ligase.
2. Competent E. coli for transformation (see Note 2) (e.g. one shot chemical TOP 10 competent cells with TOPO cloning kit): store in 200-µl aliquots at −80°C.
3. LB (Luria Bertani) medium: combine 10 g of tryptone, 5-g yeast extract and 5-g NaCl in 1 L of dH2O, Adjust pH to 7.4 using 1-M NaOH, divide into 250-ml fractions in glass bottles and autoclave.
4. LB-agar plates: add 3.75 g of bactoagar to 250 ml of LB broth in a glass bottle, autoclave to dissolve and sterilise. To prepare LB-agar plates with 100-µg/ml ampicillin heat the LB-agar in a microwave to melt, swirl the LB-agar to ensure that it is com-pletely dissolved and allow it to cool to around 37°C. Add 250 µl of 100-mg/ml stock ampicillin and pour into 10-cm disposable Petri dishes, using the aseptic technique. Flame the surface of agar plates with a Bunsen burner to remove bubbles, cover and allow to set. Store the plates inverted and wrapped in nescofilm at 4°C for up to 2 weeks. Before use pre-warm to 37°C by invert-ing the Petri dish and placinvert-ing it in a 37°C incubator, removinvert-ing the condensate from the lid.
3.2.3. Subcloning, Plasmid Amplification and Analysis of Splice Variant Amplicons
5. Ampicillin: prepare stock solution of 100 mg/ml in sterile dH2O.
Store in 0.5-ml aliquots at −20°C. Use at a final concentration of 100 µg/ml.
6. DNA miniprep plasmid purification: QIAprep spin miniprep kit (Qiagen, Cat. no. 27104).
1. As pre-mRNA splicing is dynamically regulated, the choice of cell type, culture conditions, tissue type and physiological status of the host animal can all determine the splice variants identified.
2. The procedures given below are designed to allow parallel processing of multiple cell culture samples (e.g. from 6-well plates) or multiple tissues.
The procedures are optimised for handling < 25 mg of tissue or 1 × 105−1 × 106 cells to give < 50-µg total RNA. The tissues/cell lines contain different amounts of RNA, so pilot experiments with the system of interest to determine RNA content are useful in avoiding overloading of the RNA extraction method that can lead to RNA degradation and inefficient cDNA representation.
1. There is no substitute for good laboratory practice. Therefore ensure that the work area is tidy and clean and that waste is disposed of promptly. The lab bench should be swabbed with 70% ethanol on a regular basis.
2. Keep all stocks in sterile containers and in aliquots sufficient for a single experiment. Sterilisation of containers and bulk buffer solu-tions is routinely performed by autoclaving. For glassware used for RNA isolation wash in RNase-free water and autoclave.
3. Clean automatic pipettes regularly and use separate sets for PCR and RNA isolation.
4. Do not share stocks/aliquots between investigators/experiments.
5. Prepare all reagents with gloved (latex or nitrile) hands and ensure that gloves are replaced frequently. Wear a lab coat that is frequently laundered.
6. Use nuclease-free pipette tips with an integral filter to minimise cross-contamination.
7. Open Eppendorf and other tubes carefully. Avoid aerosols/leaks and clean immediately. For PCR tubes a PCR closing tool is useful to avoid gloved fingers coming into contact with the lid.
8. Wherever possible separate the following activities into different zones: RNA isolation and first strand cDNA synthesis, PCR, bacteria plate and growth.