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Rnalater ice solution

Manufactured by Thermo Fisher Scientific
Sourced in United States

RNAlater-ICE solution is a laboratory reagent designed to stabilize and preserve RNA in biological samples. It is intended for use in the collection, storage, and transportation of RNA samples prior to RNA extraction and analysis.

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5 protocols using rnalater ice solution

1

Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted from brain, liver, and spleen samples (50 mg) using TRIzol reagent (Invitrogen) followed by purification with a RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. To note, the spleen samples were immersed in RNAlater-ICE solution (Thermo Fisher)>24 h prior to extraction. RNA concentration was determined spectrophotometrically at 260 nm, while the quality of the purified RNA was determined by 260 nm/280 nm ratio. All of the RNA samples were of acceptable quality having ratios between 1.9 and 2.1. Sample quality and the absence of significant degradation products were confirmed by establishing that every sample had RNA Integrity Number (RIN), measured on an Agilent Bioanalyzer, of greater than 7. cDNA was synthesized using MultiScribe RT enzyme (Applied Biosystems) under the following conditions: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, and hold at 4°C.
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2

Whole Lung RNA Extraction and Microarray

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Total RNA was extracted from whole lungs flash frozen in liquid nitrogen immediately after removal from sacrificed animals. Lung tissues were immersed in RNA-later ice solution over night at 4°C (Thermo Fisher Scientific, UK) to stabilize the mRNA populations prior to tissue homogenization.
Lung tissues were homogenized in a Tissue Lyser (Qiagen, UK) 2x 2 min at 30 rpm and centrifuged at 13.2K rpm for 3 min to remove cell debris. Total RNA was extracted using RNeasy mini columns (Qiagen, UK) with an additional step of genomic DNA removal through agDNA eliminator column.
RNA was quantified using a Nanodrop One C spectrophotometer (Labtech International) and quality checked using an RNA Screen Tape on aTape Station instrument (Agilent Technologies). All the extracted RNAs used in the subsequent analysis had an RNA integrity number (RIN e )>6. Total RNA (200ng) was labelled with Cy3-CTP using the Low input Quick Amp One Color labelling kit (Agilent Technologies) and hybridized onto whole genome 8 X 60K mouse microarrays v2 (AMADID 074809) following the manufacturer's instructions. The microarrays were washed and scanned using an Agilent microarray scanner G2505C.
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3

Dual RNA and Protein Extraction

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Lung tissues from healthy and tumor-bearing mice were harvested and immersed in RNAlater solution (Invitrogen). Alternatively, snap-frozen tissues and OCT-embedded tissues were thawed for 24 hours at −20°C in RNAlater-ICE solution (Invitrogen). RNA and protein were harvested from the same sample using a modification of the Nucleospin RNA/Protein kit (Machery-Nagel). In this modified protocol we included 0.5% antifoam solution in the sample homogenization which was performed using Y-30 using M-tubes (Miltenyi Biotech), and a GentleMACS disruptor on the “RNA” protocol (Miltenyi Biotech). These lysates were passed through a 20-ga needle at least 5 times to produce the stock lysate. Stock lysates were then processed for total RNA and protein isolation following the manufacturer’s instructions (Machery-Nagel). RNA samples were quantified and quality-checked by 280 nm absorbance and TapeStation gel electrophoresis (Agilent) before use. For total protein isolation, a final centrifugation step at 11000 x G for 1 minute was performed before the supernatant was collected, quantified using the Protein Quantification Assay (Machery Nagel), and aliquoted.
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4

Quantitative Analysis of AGER Expression in Human Lung Tissue

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Around 50–75 mg of whole human lung tissues were pre-treated with RNAlater-ICE Solution (Invitrogen, Life Technologies, Carlsbad, CA, USA, AM7030) and processed with a Polytron tissue homogenizer. RNA was then extracted according to the TRIzol protocol (TRIzol, Life Technologies, Carlsbad, CA, USA). Using the Nanodrop 2000c (Thermo Scientific), RNA concentrations were determined spectrophotometrically, and all samples underwent RNA quality control (RIN > 6). To generate the first-strand cDNA, the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR was used according to the manufacturer’s instructions (Invitrogen by Life Technologies, Carlsbad, CA, USA, #11752). The quantitative PCR was run in triplicates using the TaqMan Universal Master Mix II (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania, #4440040). TaqMan primers for AGER (Life Technologies, Carlsbad, CA, USA, Cat#Hs00542584_g1) and GAPDH (housekeeper; Life Technologies, Carlsbad, CA, USA, Cat#Hs02758991_g1) were used.
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5

RNA Extraction from HCC Cells and Tissues

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Total RNA was extracted from HCC cell lines HepG2 and Huh7 and immortalized normal liver PH5CH cells using Qiagen miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was also extracted from two Grade-2 HCC tumor tissues (HCC103T, HCC105T, BioChemed) and a normal liver tissue using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). All kits were used by following manufacturer guidelines. Tumor samples corresponding to two plasma samples (HCC103P, HCC105P, BioChemed) were investigated by RNAseq and one of them (HCC103T) was also studied by RT-qPCR. 100 mg tissue treated with RNAlater-ICE solution (Invitrogen, Carlsbad, CA, USA) was homogenized in a glass homogenizer, worked-up following kit protocol, and RNA eluted in 100 μl RNase-free water. Plasma (3 ml) from HCC101P (BioChemed) was spun at 2,000g for 15 minutes to remove cellular debris and supernatant was collected. Total RNA from extracellular vesicles (EVs) was extracted using the ExoMir Kit (BIOO Scientific Corp., Austin, TX, USA). RNA from the remaining EV-free flow-through plasma was extracted using the Qiagen RNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). RNA from plasma EVs, EV-free plasma, and liver tissues was sequenced at TJU as described above.
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