DOI: 10.5061/dryad.vt4b8gtvp
title: Reference genes for quantitative Arabidopsis single molecule RNA fluorescence in situ hybridization

description: Abstract Subcellular mRNA quantities and spatial distributions are
 fundamental for driving gene regulatory programmes. Single molecule RNA
 fluorescence in situ hybridization (smFISH) uses fluorescent probes to
 label individual mRNA molecules, thereby facilitating both localization
 and quantitative studies. Validated reference mRNAs function as positive
 controls and are required for calibration. Here we present selection
 criteria for the first set of Arabidopsis smFISH reference genes.
 Following sequence and transcript data assessments, four mRNA probe sets
 were selected for imaging. Transcript counts per cell, correlations with
 cell size, and corrected fluorescence intensities were all calculated for
 comparison. In addition to validating reference probe sets, we present
 sample preparation steps that can retain green fluorescent protein
 fluorescence, thereby providing a method for simultaneous RNA and protein
 detection. In summary, our reference gene analyses, modified protocol, and
 simplified quantification method together provide a firm foundation for
 future quantitative single molecule RNA studies in Arabidopsis root apical
 meristem cells.
description: Single-molecule RNA FISH probes conjugated with Quasar 670 dye
 were designed to detect <em>MON1</em>
 (<em>AT2G28390</em>), <em>TIP41</em> (<em>AT4G34270</em>), <em>AP2M</em> (<em>AT4G35800</em>), <em>NRPB1</em> (<em>AT4G35800</em>) and <em>PP2A</em> (<em>AT1G13320</em>) mRNA. These probes were applied to <em>Arabidopsis</em> root meristem cells of 5-day-old Columbia (Col-0) seedlings. Nuclei were also stained with DAPI. Protocol details can be found here: https://www.protocols.io/view/modified-arabidopsis-root-smrna-fish-protocol-rm7vzyworlx1/v1 Details of the microscope setup used to acquire these raw images: Microscope model and manufacturer Zeiss Elyra PS1 inverted wide-field microscope Acquisition software Zen 2.3 (Black) Objective Plan-Apochromat 100x/NA 1.46 DIC Immersion Oil Camera Andor iXon 897 (512x512, QE&gt;90%) Voxel Size x/y 100 nm, z 200 nm Frame Averaging 1 Acquisition  Complete z-stacks were acquired for Q670 probes then DAPI, sequentially. Q670 probe detection laser 642 nm laser diode Q670 probe emission bandwidth LP655 nm Q670 probe exposure time 1000 ms Q670 probe EM Gain 24 DAPI detection laser 405 nm laser diode DAPI emission bandwidth 420-480 nm DAPI exposure time 300 ms DAPI EM Gain 25 Probe sets used as part of this study are listed below: <strong><em>AT2G28390 (MON1)</em></strong> <strong><em>AT4G34270 (TIP41)</em></strong> <strong><em>AT4G35800 (NRPB1)</em></strong> <strong><em>AT5G46630 (AP2M)</em></strong> <strong><em>AT1G13320 (PP2A)</em></strong> ggagaagacctcgaatctga cggactatccaaccaagac aacggaaacctcgtatccat tcgacgacggagcaaaggaa ccgagcgatctatcaatcag cgaattcggtgtcggatgat gatgtcacttgtgtgacca tccgatcaatcgtaccaaga tgagatttgaggttcgacga gacatcctcaccaaaactca atcggagctaggatttggat cactaccttttctcctccg agccatattagccatacatg gacgatctggttacaggaga tcgggtataaaggctcatca ttgaacacgctccgaattcg tctgaatcgccggctaata gcttagcgagctcaagatag tcgccacggagattcagaaa tagctcgtcgataagcacag agacttcgctaggttgagat ctttatcgaccaccgtctc ctgcttaaacttatgctcct cggtaggtgcgatttatgag ccaagagcacgagcaatgat gatgcaacctcatcatcatt ccccggaagacttcagaac gcctattcttaggattcttg aaatgcatccaccatgtttc atcaactcttttcttgtcct ccacaccttcacgcaataaa gacgtccgtcgggaagaag gctcttttttactggttcat tcgtctgcattatatgcgta catcgtcattgttctcacta cttaacaaaacctctcctcc agtcatggatgcgtaaacc accctcaatagtcagttttg cacaggacaattccctaact atagccaaaagcacctcatc cttccatcatcagctttata cgcggagggtttcgatctc tgacaatccgcgtcactaat taaacaaaggagcagccacc atacagaataaaacccccca attagcttccctaatctcat cttcgacagcgagagaagt aggacgagcaaacttagggt tgtagacattgctgattcgc caagtttcctcaacagtgga ctcatcaacatggcgttttc tcttaagctttttttccca gggtggaggaataggaagga gcaacattagcattgctact tcatctgagcaccaattcta tccatgacgtagaagcatca ccatttcaggtaagtgaga ctgccttttcaagttttcat agctacagcctcaacaacaa tagccagaggagtgaaatgc tgcctgagttactcagtatg gactaatgcattctcgcca gtgccgagaaatcaacacgt gctccaccaaagtatgattt cattcaccagctgaaagtcg gttcatctccatatctggaa gtgttgctacctaagtgtt cagagcaatactccacggaa ttctaatagcgtcttcatca ggaaaatcccacatgctgat gctgaaaatccagcaagctt accagctagtgcgtcaaac gtccataatcaacaagctcc agagattttgtggatagcca atattgatcttagctccgtc accaccattctccacaaaag caacaggtggaagcccttc ctggggggagacaatacatt acaccttcctgagtgatata attggcatgtcatcttgaca ccttgactaagttgacacgg attgtgctgcagcaggaac agaggacgaggcttcaagat gtcttttggcttcgatgaaa aaattagttgctgcagctct aagacaacctggtgatttcc acggcttgctcctgaattt gcagagtacctcaacagatt atgttgcatttggaacaggt gctgattcaattgtagcagc agatatattggccccttaac aatctagtatcacctgctg gcaaagagttccggcaagaa caaccaacagcacctgtaac ccgaatcttgatcatcttgc tgtttcatctgtacagctga ggcgttgtaaacgtgtaat tccattagatgtaccaaggg ttctttttgtaagcaaggcc caaccctcaacagccaataa tatagaagatccaactgccc actacttcacttccacagt aacctcttcccaaatgacat cactttccacaatatccaga ctccaacaatttcccaagag gcaagggtgtcatatcgaac accgtttctttgtctttct cgaggaattttctagctgca ttgcctttagaggacatcag caaccatataacgcacacgc tgagaagacagcatctgtcc tttggattagcttttgcct aattgtgtcaccaattccga cgtcacatcacaccgaagaa agtagacgagcatatgcagg ctgggttccagctaaatgaa cactgaagagtagcttccc actgccggataagatctttc aggcattcccgaaaggaaac gaacttctgcctcattatca ccttaacgcatatggaaggg atctgatcttcacagttct tggttaaccctgttctcaaa cattcaaccccaacttcaaa cacagggaagaatgtgctgg acttcttgcaatatggttcc ttaagtgagagagcagcca gcatcatcacgagctttatt tccttctcaagaccaatttt tgacgtgctgagaagagtct gaataagacaccagacgcgc gccagttcatcttcataca caacattttgctgaccgaca tgggcgagatttcatttctg cccattataactgatgccaa tgtgtctgcacattagtagt cagtcagaagtgacactcc tttcaaggaatccagcttct ctcaatggttttaccactct tggttcacttggtcaagttt gtagaagcaagtcatcggga cttggcatgactctcactt cccttcaagtcttcaagatg caggttcacacactggtgaa tctacaatggctggcagtaa tgagcgttgtatcttggtag tcgcaagaggaggaaccat cgagttggaatctgtcagtc acagtcttctcggagttgaa cgattatagccagacgtact agaagtggacataggcatgc gcacaccatcaactctcag caagtgctatcaccatttgt gatttacaccctctgttatt gactggccaacaagggaata gaacgcatctgaacgtgtgg ctcgtctctctcaatctca cagatatgcctcttgatgtt gtgtacgaccaagttctttg catcaaagaagcctacacct taccctgcaatctttgagat tcgccaaacctataatgca tcaacagggtgcatatctga accttgacatttacttccat ttgcatgcaaagagcaccaa cgcgattgatctttgaacca cgaagaacagttggtgcct cacactcaacgcatcatcac agggcaaacattttagcacc acggattgagtgaaccttgt ggtacatcttcaacacgcat atgttgcttctctccaaca attgagcagcaacacaaccg tggaattttaaccacgaccc cttcagattgtttgcagcag tagtagatgatcgacgcctg taccctttcgcagatagag cgtaacgttctttgcactga cttggaaattggtcttggct ggaccaaactcttcagcaag ggtgagtcttgttcttgatt ctccagactgctaagtcaa tgttttgatcctcttagcta tggattgtatttagcgcgac ggaactatatgctgcattgc atgccaaagtccaaagggac tgactgatggagctcgggt ccggagtgagatagactgat cttagcgtggactctgtttg gtgggttgttaatcatctct gttactgggggtgagaattc tgcttaatcactggaagcc acatcttcatctggcatttc ccattgttgagatcaactca tgcacgaagaatcgtcatcc gacttttctgttgtctgtga gggatcttcagtttctgtg aagtagccacggagatatct cttgtccaagatttcttctc ttactggagcgagaagcgat agtttctggtatgctcgata aaagctcatcggtacgctc catctcgcgattcaactcta tgtgaacattggcacctgaa gaacatgtgatctcggatcc ccaatccttttacatgcatt agtagtttgtttggctgta gatgtctggaatacctctga aacgaactcgcagaccagat ctctgtctttagatgcagtt gtgtagttttcatctcttct agtcagtggagtaactcct agcaaagcacgacgaactgc ttatgtatcgaacccactcg catcattttggccacgttaa tggtgtgacccaacatagaa gttcatatgaaaccaccac cgagtgatagccatcagatg cagcatctgatctcgtaaga cgtatcatgttctccacaac gcaagtggatcaaatgctgc   tcttcagcatctcatcattc   atcaacatctgggtcttcac cacacctgattgcatatctt   atagctagggagctgaagtt   ttggagagcttgatttgcga cttcattctccacatctttt   tgacactggtgaacgagcag   acacaattcgttgctgtctt aaaggactagctccttgcaa   catcatgccttcatggtaag   cgcccaacgaacaaatcaca
description: ImageJ