Computational protocol: New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

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Protocol publication

[…] In the current communication, master mix and reverse transcriptase (RT) enzyme are used as suggested and provided in a commercially available kit (Cat. No. 4309169, TaqMan® One-Step RT-PCR Master Mix Reagents Kit, ABI). The Multiscribe™ (MuLV) RT enzyme (10 U/μl) included in the kit arrives already pre-mixed with RNAse inhibitor (40 U/μl, ABI) as a 40X solution. The one-step master mix (which contains AmpliTaq Gold hot-start DNA Polymerase, 5.5 mM MgCl2, 10 mM of each A, C and G dNTP’s and 20 mM dUTP, ROX passive internal reference molecule, a PCR product carryover correction component, other ABI-proprietary buffer components, but no AmpErase UNG enzyme) arrives as a separate 2X solution (5 ml total) in the kit. Each of our 30 μl one-step real-time RT-PCR reactions contain: 15 μl one-step Master Mix, 0.25 U/μl Multiscribe™ RT enzyme, 0.4 U/μl RNAse inhibitor, optimal forward and reverse primer and fluorogenic probe concentrations (previously established for each target using two-step real-time RT-PCR according to classic ABI protocol, see below), nuclease-free water, and 7.8 μl of each LCM-[RNA template] cell extract isolate. Before use, all solutions are gently vortexed and spun down. Our thermocycling conditions for fluorogenic one-step RT-PCR are: 35 minutes at 48°C (for reverse transcription; normally 30 minutes; ABI), 10 minutes at 95°C (for AmpliTaq Gold DNA polymerase hot-start activation), and 50 cycles of: 15 seconds at 95°C (for duplex melting), 1 minute at 58°C (for annealing and extension; normally 60°C; ABI). [Note: we altered the ABI-suggested thermocycling conditions for their one-step master mix in favor of what we found to be more optimal for our particular targets]. For recent real-time results using this IF-IHC-LCM-RT-PCR method, see Figures -. In addition, Figure shows that we have recently detected one-step real-time RT-PCR signal in samples containing RNA from the equivalent of a single cell (using the common assumption that one mammalian cell contains 10-30 pg of total RNA, but upon isolation, yields ~10 pg of RNA).We abandoned the use of 18S Ribosomal RNA as housekeeper (see also Appendix A, item 6), since the primers and probe provided as part of an ABI RNA control reagents kit were of a faulty design (). Instead, we used ovine ribosomal protein S15 (ovRPS15) as the housekeeping (endogenous reference) gene, the sequence of which we received from Dr. Sean Limesand (Department of Pediatrics University of Colorado Health Sciences Center, Perinatal Research Center, PO Box 6508, F441, Aurora, Colorado 80045), which he had successfully cloned into a pCRII plasmid and used as a housekeeping gene with conventional PCR work in sheep pancreas:ovRPS15 in pCRII as read from the T7 promoter: 5’-ttccgcaagt tcacctaccg cggcgtagac ctcgaccagc tgctggacat gtcctatgag caactgatgc agctatacag cgcgcgcca gcgacggcgg ctgaaccgcg gcctgcggag gaagcagcac tcgttgctga agcggctgcg caaggccaag aaagatgcgc cgcccatgga gaagcccgag gtggtgaaga cgcacctgcg cgacatgatc attctgcccg agatggtggg cagcatggcc ggcgtctaca acggcaagac cttcaaccag gtggaaatca agcctgagat gattggccac tacctaggcg agttctccat cacctacaa gcccgtaaag catggccggc ccg-3’.We designed real-time primers and probe to this ovRPS15 sequence using computer software from ABI (Prism Primer Express v. 2.0). Our sequences for the forward and reverse primers and fluorogenic probe for ovRPS15 are: 5’- CGAGATGGTGGGCAGCAT-3’, 5’-GCTTGATTTCCACCTGGTTGA-3’, and 5’-VIC-CCGGCGTCTACAACGGCAAGACC-TAMRA-3’ (where VIC™ is a proprietary fluorescent reporter dye known only by its 3-letter acronym; a trademark of Applera Corporation). We also designed sequence-specific oligonucleotide primers and fluorescent probes for our other real-time targets of interest (SBD-1, SP-A, SP-D, ovine MCP-1α and TLR4) using Primer Express v.2.0 in conjunction with the search tool BLAST (Basic Local Alignment Search Tool, National Center for Biotechnology Information). Resultant probe sequences are checked for specificity by comparing them for similarity to all other available sequences in the database. Only unique sequences and/or sequences that spanned genomic introns are used for our SP-A, SP-D, SBD-1, MCP-1α and TLR4 primer and probe designs. We identified ovine TLR4 real-time primers and probe by trial and error using the bovine sequence for TLR4 (accession number NM 174198) as a general ruminant TLR4 template. The second real-time primer-probe set we designed for ovine TLR4 (and tested on ovine lung total cDNA) worked beautifully. The forward and reverse primers and fluorogenic probe for “ovine” TLR4 is as follows: 5’-GAGAAGACTCAGAAAAGCCTTGCT-3’, 5’-GCGGGTTGGTTTCTGCAT-3’ and 5’-6FAM-TAAACCCCAG AGTCCAGAAG GAACAGCA-3’, respectively. The primer and probe sequences for our other targets are: for SP-A, 5’-TGACCCTTATGCTCCTCTGGAT-3’, 5’-GGGCTTCCAAGACAAACTTCCT-3’, and 5’-6FAM-TGGCTTCTGGCCTCGAGTGCG-TAMRA-3’; for SP-D, 5’-acgttctgcagctgagaat-3’, 5’-tcggtcatgctcaggaaagc-3’, and 5’-6FAM-ttgactcagc tggccacagc ccagaaca-TAMRA-3’; for SBD-1, 5’-CCATAGGAATAAAGGCGTCTGTGT-3’, 5’-CGCGACAGGTGCCAATCT-3’, and 5’-6FAM-CCGAGCAGGT GCCCTAGACA CATGA-TAMRA-3’ and for ovine MCP-1α (the sequence used for ovine MCP-1α primer and probe design: 5’-GAGAGGGGCC AATCCAGAGG CCAACAGCTC CCACGCTGAA GCTTGAATCC TCTCGCTGCA ACATGAAGTT CTCCGCTGCT CTCCTCTGCC TGCTACTCACA GTAGCTGCCT TCAGCACCGA GGTGCTCGCT CAGCCAGATG CAATTAACTC CCAAATTGCC TGCTGCTATA AATTCAATAA GAAGATCCCC ATACAGAGGC TGACAAACTA CAGAAGAGTC ACCACCAGCA AGTGTCCCAA AGAAGCTGTG ATTTTCAAGA CCATCCTGGG CAAAGAGTTT TGTGCAGACC CCAACCTGAA ATGGGTCCAG GACGCCATAA ACCATCTCAA CAAGAAAAAC CAAACTCCGA AGCCTTGA-3’ was provided in a recent publication ()): 5’-GCTGTGATTTTCAAGACCATCCT-3’, 5’-GGCGTCCTGGACCCATTT-3’, and 5’-6FAM-AAAGAGTTTT GTGCAGACCC CAACC-TAMRA-3’; (where 6FAM is 6-carboxyfluorescein, the fluorescent reporter dye, and TAMRA is 6-carboxytetra-methylrhodamine, the fluorescent quencher dye). We determined our optimal real-time concentrations for target and endogenous reference forward and reverse primers using the standard grid testing method as suggested by ABI (see "Additional Details Regarding Optimizing Real-Time Primers and Fluorogenic Probes Before their use with Experimental Samples: Optimization and validation tests performed on cDNA" section below for more details). Our optimal concentrations were: 300 and 300 nM for ovine SP-A, 300 and 300 nM for ovine SP-D, 300 and 900 nM for SBD-1 forward and reverse primers (), 900 and 400 nM for ovine MCP-1α and 1000 and 1000 nM both for ovine TLR4 and ovRPS15 forward and reverse primers, respectively. Similarly, optimal concentrations of fluorogenic probes are found to be 50 nM for SP-A, 100 nM for SP-D, 150 nM for SBD-1, 100 nM for TLR4, 100 nM for ovine MCP-1α and 150 nM for ovRPS15. Each plate contained both target and endogenous references for all samples present on that plate, and a negative no-template control ("NTC"; nuclease-free water) for each target and endogenous reference. No reverse transcriptase controls or "NRC" (one-step reactions containing everything except reverse transcriptase enzyme to test for genomic DNA contamination) are run on each RNA sample in separate plates prior the actual sample plates. Duplicate replicates are run for each of these time-intensive LCM samples. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Application qPCR