Qubit rna hs kit

Fresh‐frozen tissue samples were collected for RNA extraction. Tissue scrolls were sectioned from OCT‐embedded fresh‐frozen samples into Buffer RLT with β‐mercaptoethanol (Qiagen Sciences LLC, Germantown, MD, USA). RNA was purified following the protocol from RNeasy Micro Kit (Qiagen). RNA concentrations were determined using Qubit RNA HS kit (Thermo Fisher, Waltham, MA, USA). RNA integrity numbers (RIN) were calculated using the RNA 6000 Pico Kit 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Total RNA was isolated from FFPE sections using the ReliaPrep Total RNA Miniprep system (Promega), and RNA concentrations were measured with the Qubit RNA HS kit (Thermo Fisher Scientific, Waltham, MA). Then, 250 ng RNA input was used for the preparation of cDNA. For preparing open-ended target enriched NGS libraries, anchored multiplex PCR (AMP) technology was applied by using the FusionPlex® kit v1, according to the manufacturer’s instructions (Invitae, San Francisco, CA). A targeted gene panel has been deployed (custom designed), including genes relevant for the differential diagnosis of sarcomas. Library preparation was done according to the manufacturer’s instructions. Pooled libraries were sequenced on a NextSeq 500 (Illumina). Demultiplexing was performed using an in-house bioinformatic workflow, and data were thereafter analyzed using Archer Analysis software (ArcherDX) version 6. All reported analyses were of good quality, defined by the following criteria: > 50% RNA reads and on average ≥ 10 start sites per gene-specific primer targeted to housekeeping genes (SS/GSP2). The reported fusion transcripts were found in the “strong fusion” section of Archer Analysis.

Total RNA was purified using the RNeasy Plus Mini Kit (Qiagen, Copenhagen, Denmark). The RNA concentration was measured by the Qubit RNA HS kit and the Qubit 4 Fluorometer (ThermoFisher Scientific) and stored at −80 °C until use. Two μg total RNA was reverse transcribed using the RevertAid Minus First strand cDNA synthesis kit (ThermoFisher Scientific) using oligo(dT) primers, following the manufacturer’s instructions. qPCR was performed with TaqMan Fast Advanced Master Mix TaqMan assays for SOX2 (Hs01053049_s1), NANOG (Hs04260366_g1), POU5F1 (Hs0099632_g1), CD44 (Hs01075864_m1), PROM1 (Hs01009250_m1), BAD (Hs00188930_m1), BAK1 (Hs00832876_g1), BAX (Hs00180269_m1), BCL2 (Hs00608023_m1), TP53 (Hs01034249_m1), CDKN1A (Hs00355782_m1), HPRT1 (Hs02800695_m1), and GAPDH (Hs99999905_m1), all from ThermoFisher Scientific. Ten ng cDNA was used per reaction, as recommended by the manufacturer’s protocols. The qPCR was performed in a QuantStudio 3 (ThermoFisher Scientific) for 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles at 95 °C for 1 sec and 60 °C for 20 s. Expression levels were normalized to HPRT1 and GAPDH, and relative expression levels were calculated using the Pfaffl-method [54 (link)].

Total RNA was isolated with a Purelink RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA integrity was analyzed with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA) obtaining RNA integrity numbers (RIN) above 8 for all samples (on a scale based on an rRNA 28S/18S ratio where a RIN of 1 corresponds to a totally degraded RNA and 10 to a totally non-degraded RNA). RNA concentration was quantified by spectrophotometry (Nanodrop, Thermo Fisher Scientific). One hundred nanograms of total RNA was reverse-transcribed into cDNA, then transcribed to cRNA and Cy3-labeled with a Low Input Quick Amp-One Color Labeling Kit (Agilent Technologies). The labeled cRNA was purified with an illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, UK) and the total yield was measured with a Qubit RNA HS Kit (Thermo Fisher Scientific). Hybridization, washing, assembling of the chips, and scanning were performed according to the manufacturers’ instructions. Briefly, labeled samples were hybridized with SurePrint G3 Human GE 8x60K chips for 17 h at 60 °C in an Agilent hybridization oven at 10× rpm. Posterior washing, stabilization and drying procedures were performed according to Agilent’s Low Input Quick Amp Labeling Kit instructions [10 (link)].

Total RNA was extracted using RNeasy Micro Kit (74004, Qiagen) according to the manufacturer’s instructions. Then, RNA quantitative detection was conducted with Qubit RNA HS Kit (Q32855; Thermo Fisher Scientific). Agilent Bioanalyzer 2100 (Agilent) was used to check the integrity of the extracted total RNA. Only samples whose RNA integrity number (RIN) is greater than 7 were considered qualified samples and used for subsequent testing.
Next, RNA reverse transcription and amplification were conducted using MALBAC^® Platinum Single Cell RNA Amplification Kit (KT110700796; Xukang Co., Ltd.). The positive and negative controls were 500 ng of high-quality host total RNA and ultrapure water, respectively. After that, 1 µl cDNA was 10-fold diluted and detected with Agilent Bioanalyzer 2100 (Agilent).
Finally, the library was constructed using gene sequencing and library preparation kit (XK-038, Xukang Co., Ltd.) according to the manufacturer’s instructions. Following purification, the library was quantified with Qubit dsDNA HS kit (Q32584, Invitrogen). According to the quantitative results of Qubit, each sample was taken from 10 ng library and mixed in equal proportions, followed by Qubit quantitative detection again. Pair-end sequencing of the products was performed on the NextSeq CN500 platform (Illumina).