Samples and total RNA extraction
Total RNA was isolated from five breast cancer cell lines (MCF-7, HCC-1187, Cal51, ZR-75-1-1, and OCUB-M) using TRI-reagent (Sigma) following the manufacturer's recommendations.
Purification of total RNA and genomic DNA removal
The following were tested:
Column method, using RNeasy mini-kit (Qiagen) with on-column DNase I treatment, following the manufacturer's instructions.
Non-column methods, using DNase I treatment followed by a clean-up step. For DNase treatment 2 units of DNase I (Roche Applied Sciences) were used per μg of total RNA at 37°C for 30 minutes. The reaction was tested on total RNA dilutions of 20 μg/100 μl and 5 μg/μl (D20 and D5 respectively). Two clean-up methods were evaluated:
1- Lithium Chloride (LiCl) precipitation. D20 samples were purified using a final concentration of 2.5 M LiCl. After incubation at -20°C for 2 hours the sample was centrifuged (16,000 g) at 4°C for 20 minutes (min). The pellet was then washed with 70% ethanol before drying.
2- Extraction with phenol:chloroform:isoamyl alcohol (25:24:1, pH: 5.2, PCI). D20 and D5 samples were mixed with one volume of PCI in a Phase-Lock-Gel™ (PLG) tube (Heavy Gel, Eppendorf). After mixing, the tube was centrifuged at room temperature (RT) for 5 min. The aqueous phase was transferred to a new PLG tube and a second extraction was done using chloroform. The aqueous phase was mixed with 100% ethanol and 0.1 volumes of 7.5 M NH4Acetate, incubated at -20°C from 2 hours to overnight (ON), followed by washing with 70% ethanol.
RNA amplification
cDNA synthesis
First strand cDNA was synthesized using 1 to 5 μg of total RNA (see below). RNA was mixed with 1 μl of T7-oligo (dT) primer (100 ng/μl, Ambion) in nuclease-free water to a total volume of 8 μl and added to EndoFree RT™ enzyme (Ambion) in a 21 μl reaction following the instruction manual. The reaction was incubated at 50°C for 2 hours.
Second strand cDNA was synthesized by mixing the first strand reaction with 95 μl nuclease-free water, 15 μl KOD XL Buffer (Novagen®), 15 μl of dNTPs (final concentration 0.2 mM), 1 μl RNase H (10 U/μl, Ambion), and 3 μl of KOD XL polymerase (Novagen®). Incubation was at 37°C for 5 min, followed by 94°C for 2 min, 65°C for 1 min and 75°C for 30 min. The reaction was stopped by 15 μl of 0.1 M NaOH/2 mM EDTA and incubated at 65°C for 10 min and neutralized by 15 μl of 0.1 M HCl.
Reactions carried out without reverse transcriptase were used as negative controls.
Purification of cDNA
cDNA was purified using the following methods:
1) cDNA clean-up column (DNA clear™ kit, Ambion) using the manufacturer's instructions.
2) PCI (pH:8.2) extraction with isopropanol precipitation at room temperature (Isopropanol method): reaction volume was adjusted to 200 μl with nuclease-free water, mixed with 200 μl of PCI and transferred to a PLG tube. After centrifugation (12,000 g) at RT for 5 min, the aqueous phase was transferred to a fresh PLG tube and a separate chloroform extraction was carried out. The final aqueous phase was precipitated using 1 μl of linear acrylamide (0.1 μg/μl, Ambion), 0.5 volumes of 7.5 M NH4Acetate and two volumes of isopropanol. The mixture was incubated at RT for 10 min and centrifuged (12,000 g) at RT for 20 min. The pellet was washed with 500 μl of 75% ethanol, centrifuged for 5 min, dried, and re-suspended in nuclease free water.
3) PCI extraction with ethanol precipitation at room temperature (ethanol at RT): Ethanol was replaced for isopropanol after PCI extraction and sample was immediately centrifuged (Modified from Zhao et al, [8]).
4) PCI extraction with cold ethanol precipitation (ethanol at CT): After PCI extraction, 0.1 volumes of 7.5 M NH4Acetate were added to the aqueous phase and mixed with 2.5 volumes of pre-chilled 100% ethanol. The mixture was incubated at -20°C for 2 hours and centrifuged at 4°C for 20 min, followed by washing with pre-chilled 70% ethanol and re-suspension.
In vitro transcription to generate amplified RNA
aRNA was generated by T7 MEGAscript™ kit (Ambion) with incorporation of aminoallyl-UTP (aa-UTP) in the process. 15 μl of purified cDNA was mixed with 3 μl of aaUTP solution (50 mM, Ambion), 12 μl of ATP, CTP, GTP mix (25 mM), 2 μl of UTP solution (75 mM), 4 μl of T7 10 × reaction buffer and 4 μl of T7 enzyme mix. A ratio of 1:1 for aaUTP: UTP was used [9]. The reaction mix was incubated at 37°C for 14 hours followed by treatment with 2 μl of DNase I (2 U/μl) at 37°C for 30 minutes.
Purification of amplified RNA
The following methods were used to purify aRNA:
1) Column purification with RNeasy kit (Qiagen) following the manufacturer's instruction.
2) PCI extraction (pH: 5.2): an equal volume of PCI was added to aRNA and transferred to the PLG tube as described above. After two rounds of PCI extraction, a separate chloroform extraction step was carried out followed by precipitation with NH4Acetate and ethanol at -20°C.
3) LiCl precipitation: aRNA was precipitated with a final concentration of 2.5 M LiCl. After cold incubation at -20°C for 2 hours, the sample was precipitated and washed as described above.
4) Guanidinium Isothiocyanate-phenol or TRI-reagent™ purification (guanidinium method). After addition of 100 μl of 4 M guanidinium isothiocyanate to the aRNA sample, it was purified using the PLG tubes and phenol as described by the manufacturer (PLG manual). Alternatively 1 ml of TRI-reagent™ (Sigma) was added to each aRNA sample, mixed well and transferred to a PLG tube. After adding 200 μl of chloroform, the solution was mixed by shaking, incubated at RT for 2 min and centrifuged (12,000 g) at 4°C for 20 min. The aqueous phase was then transferred to a new PLG tube and mixed with 600 μl of chloroform. After centrifuging (12,000 g) at 4°C for 10 min, the aqueous phase was transferred to a 1.5 ml tube and precipitated by adding 1 μl of linear acrylamide (0.1 μg/μl, Ambion), 0.1 volumes of 3 M NaAcetate and an equal volume of isopropanol followed by incubation at -20°C ON. The centrifuge and washing steps were carried out as described previously for PCI extraction.
Labelling of amplified RNA
Coupling reaction
Aminoallyl modified-aRNA (aa-aRNA) was coupled with monoreactive Cy3 and Cy5 dyes (Amersham). One vial of dye was dissolved in 40 μl of dimethylsulfoxide (DMSO) and divided into aliquots of 4 μl and dried by speed vacuum. To 10 μg of aa-aRNA in 6.7 μl of nuclease-free water, 10 μl of DMSO, and 3.3 μl of 0.3 M NaHCO3 (pH: 9) were added. The mixture was immediately transferred to Cy3 or Cy5 dried dyes and mixed by pipetting. Coupling reactions were carried out for 1 hour in the dark followed by quenching with 4.5 μl of 4 M hydroxylamine for 15 minutes.
Purification of labelled aRNA
Labelled targets were cleaned-up by the following methods:
1) Column purification with Qiagen RNA columns.
2) LiCl-Ethanol precipitation was carried out by adding 0.1 volumes of 4 M LiCl and 2.5 volumes of pre-chilled 100% ethanol. The mix was incubated at -20°C for 2 hours and centrifuged (12,000 g) at 4°C for 20 min followed by washing with 500 μl of pre-chilled 70% ethanol and re-spinning at 12,000 g for 5 min. The pellet was then air-dried and re-suspended in nuclease-free water.
3) PCI extraction (pH: 5.2): One round of PCI extraction followed by precipitation.
4) LiCl precipitation. To each reaction 12.5 μl of 7.5 M LiCl was added (2.5 M final concentration). The mixture was incubated at -20°C overnight followed by precipitation as described before.
Assessment of RNA quality
The NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies) was used to determine: 260/280 ratio, to assess total RNA and aRNA purity; 320/550 and 320/650 ratios, to evaluate Cy3 and Cy5-labelled aRNAs respectively.
Quality of total RNA and patterns of amplified or labelled aRNA were evaluated using the Agilent-2100 Bioanalyzer with the RNA 6000 Nano Lapchip® kit (Agilent Technologies) and also by 1% denaturing agarose/formamide gels. Coupling efficiency for Cy5 dyes was assessed using the Agilent Bioanalyzer.
Hybridisation of cDNA microarrays
Expression microarrays containing 6528 pairs of duplicate cDNA spots were used (Cancer Research UK DNA Microarray Facility at the Institute of Cancer Research; CR-UK DMF Human 6.5 k genome-wide array).
Labelled targets from two cell lines, Cal51 and ZR-75-1-1, were generated using the optimized purification protocol. A total of 4 hybridizations were done: two slides were used with the same dye combination (replicates) and two slides with reversal of the dyes (dye reversal).
For each hybridization 2 μg of each Cy3 and Cy5-labelled targets (corresponding to 110–130 pmols of dye) were used. Hybridisation was performed as described http://www.crcdmf.icr.ac.uk with minor modifications. In brief, the volume was adjusted to 15 μl with nuclease-free water, to which 15 μl of pre-warmed (37°C) Amersham Hybridisation Buffer (Amersham Biosciences), 30 μl of deionised formamide, and 1 μl of Poly-dA (10 μg/μl, Amersham Biosciences) were added. After mixing, the samples were denatured at 92°C for 2 min and centrifuged at 12,000 g for 5 min. Slides were placed in Glass Array Hybridisation Cassettes (Ambion), targets were applied and cover slips fitted. Hybridization was carried out at 42°C overnight in a waterbath.
Washing was done in 2XSSC, 0.2%SDS at 42°C for 30 min, 2XSSC, 0.1%SDS at 42°C for 30 min, and 0.1XSSC, 0.1%SDS at RT for 10 min. Slides were then plunged ten times in 0.1XSSC to remove extra SDS with subsequent washes in 0.1XSSC two times for 2 minutes and once for one minute. Subsequently they were washed in 0.01XSCC for 15 seconds and submerged quickly in 96% ethanol followed by spin-drying at 500 rpm for 5 min.
Scanning, feature extraction and analysis
Slides were scanned using the ScanArray® 4000 microarray analysis system (Packard BioChip Technologies). Feature extraction was done using ScanArray Express software (Packard BioChip Technologies) and spots with high background were flagged manually. Data was transferred as tab delimited text files and analyzed using R mathematical program http://cran.us.r-project.org and Statistics for Microarray Analysis package (SMA), http://stat-www.berkeley.edu/users/terry/zarray/Software/smacode.html. Student's t-test and Chi-Square statistics were used for analysis of parametric and non-parametric factors respectively.
Bioinformatics
The longest annotated transcript for each of the analysed genes was computationally determined after combining the information available in RefSeq [http://www.ncbi.nlm.nih.gov/RefSeq/ and http://ncbi.nlm.nih.gov/books/bookres.fcgi/handbook/ch18d1.pdf, The NCBI handbook, Chapter 18. The Reference Sequence (RefSeq) Project] and Ensembl (Human v.16.33.1, http://www.ensembl.org) databases [15]. The search was automated by using BioPerl http://www.bioperl.org and Ensembl Perl modules on a Linux platform [16].
Reverse Transcription-PCR of selected transcripts
RT-PCR was done using RNA from cell line HCC-1187 to amplify a 50 base pair (bp) fragment of R38bsno and a 7130 bp fragment of Human guanine nucleotide exchange factor (p532) located 1 kb from the 3' end of the cDNA.
Reverse transcription was done using either 5 μg of total RNA with 1 μl of oligo-dT (16) primer (Roche Applied Biosciences) or 2 μg of aRNA with 25 pmols of gene specific reverse primer. The volume was adjusted to 9 μl with nuclease-free water, incubated at 70°C for 3 min and cooled on ice for 2 min. The following were added to the primed RNA: 4 μl of first strand buffer (BD Biosciences Clontech), 2 μl of 0.1 M DTT, 1 μl of RNase Inhibitor, 2 μl of 10 mM dNTPs, and 2 μl Powerscript Reverse™ Transcriptase (BD Biosciences Clontech). The reaction was incubated at 42°C for 2 hours, heat inactivated for 15 min at 70°C, and treated with 1 μl of Ribonuclease H (Promega) at 37°C for 30 min.
The primers used for PCR amplification were (p532 primers designed as described [17]):
R38bForward-5'GCTGAGTCCATGATGATTTC3'
R38bReverse-5'GCCTTTCTTTGCCTTCAGAC3'
p532Forward-5'AACTCACGGCAGTGGAGGGAAAG3'
p532Reverse-5'TGCTGTTCTGGTTGTTGGGGCTA3'
PCR conditions were:
For R38b-2 μl of reverse transcription product, 8 μl of MgCl2 (25 mM), 4 μl of 10 × PCR Buffer2 (Promega), 2 μl of 10 mM dNTPs, 1 pmol of each of the primers, and 1 μl of AmpliTaq polymerase (Promega). The volume was adjusted to 50 μl and thermal cycling carried out for 1 min at 95°C, 45 seconds at 51°C, 1 min at 72°C for 30 cycles with a 10 min extension period at 72°C in the last cycle. As a negative control, cDNA was excluded from the PCR reaction. The product was analysed on a 2.5% agarose gel.
For p532-2 μl of reverse transcription product, 2 μl of each of the primers (5 pmol/μl), and KOD XL Polymerase (Novagen®) following the product instructions. Thermal cycling was carried out for 10 seconds at 94°C and 7.1 minutes at 68°C for 35 cycles, followed by 10 minutes at 72°C. Negative control included a PCR reaction without cDNA. The PCR product was analysed on a 0.7% agarose gel.