Rneasy mini kit rna cleanup protocol

Total RNA was isolated using a previously described protocol (7 (link)) for the PureLink FFPE total RNA isolation kit (Invitrogen). Briefly, cells were sorted into the melting buffer containing 1600 U/mL RNase inhibitor (Bioline) and 10 mM DTT (Sigma-Aldrich Ltd.) and stored at −80°C before proceeding to the proteinase K treatment for 15 min at 60°C. Subsequently the manufacturers instructions were followed, including the optional DNase digestion. The RNA was further cleaned using the RNeasy Mini Kit RNA Cleanup protocol (Qiagen). RNA concentrations were measured using the NanoDrop 2000 (Thermo Scientific) and RNA integrity assessed using the 2100 Bioanalyser instrument (Agilent Technologies, Inc.).

Total RNA was isolated from cells grown to full confluency in 50-mm cell culture dishes using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Approximately 1 × 10⁶ cells were washed once with 5 ml PBS. TRIzol (Invitrogen) was added directly to cells, vortexed for 20 s and incubated at RT for 5 min. 200 µl chloroform was added to each tube and vortexed for 10 s. The sample was incubated at RT for a further 3 min and centrifuged for 15 min at 13,200 rpm (4 °C) to separate the aqueous and organic phases. The upper, aqueous phase containing the RNA was transferred to a new 1.5-ml microcentrifuge tube and placed on ice to precipitate with ice-cold isopropanol for 15 min. Precipitated RNA was pelleted by centrifugation for 10 min at 13,200 rpm (4 °C) and washed in 200 µl ice-cold 75% ethanol. The pellet was resuspended in ice-cold nuclease-free water and subject to RNase-Free DNase Set (Qiagen, cat. no. 79254) according to the manufacturer’s instructions. RNA was prepared using RNeasy Mini Kit RNA Cleanup protocol (Qiagen), and the concentration was determined using a Qubit QuantIt RNA quantitation kit (Invitrogen). Samples were stored at -80 °C or used directly in next-generation sequencing library preparation.

We generated a custom in situ probe library by using RT-PCR to amplify marker genes of interest from total RNA isolated and pooled from zebrafish embryos between 1–6 dpf (see Supplementary file 1 for genes, probe lengths, and RT-PCR primer pairs). PCR fragments were ligated into a pCRII vector using the TOPO TA Cloning Kit (Invitrogen). Digoxigenin-labeled RNA probes were transcribed from linearized template using a SP6/T7 DIG RNA Labeling Kit (Roche, Basel, Switzerland) and purified using the RNeasy Mini Kit RNA cleanup protocol (Qiagen, Hilden, Germany).
Whole-mount in situ hybridization (WISH) was performed essentially as described (Thisse and Thisse, 2008 (link)), but with several important modifications that minimize physical distortions to fixed embryos during high temperature hybridization steps, optimize the robustness of the staining reaction, and increase the stability of stained samples during clearing and tomographic imaging steps. All modifications from the standard zebrafish WISH protocol (ZFIN Protocol Wiki, Thisse Lab 2010 update) are included in the summary below: