Reliaprep ffpe total rna miniprep kit

RNA was extracted from FFPE slides of 21 patients using the ReliaPrep™ FFPE Total RNA Miniprep kit (Promega, Madison, WI, USA). Extracted RNA was then synthesized into cDNA using the TruSeq RNA Exome Kit (Illumina, San Diego, CA, USA), and an RNA sequencing dataset was generated using the Nova 6000 platform (Illumina). The raw data were quantified using the STAR [19 (link)] and RSEM [20 (link)] quantification tools, and differentially expressed gene (DEG) analysis was performed using DESeq2 [21 (link)]. The threshold for identifying DEGs was adjusted to absolute log2 fold change ≥ 1 and p < 0.05. The DEGs were plotted in a volcano plot graph using the “ggplot” package in R.

Libraries were prepared for 50 bp single-end sequencing using a NEXTflex Small RNA-Seq Kit (PerkinElmer Inc., Waltham, MA, USA). Total RNA was extracted from FFPE samples using the ReliaPrep FFPE Total RNA MiniPrep Kit (Promega, Madison, WI, USA). Small RNAs were synthesized as single-stranded copy DNAs (cDNAs) using reverse transcription priming. The quality of these cDNA libraries was evaluated using a TapeStation 4200 (Agilent, Santa Clara, CA, USA), followed by quantification using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s protocol. Sequencing was progressed as single-end (50 bp) using an Illumina NovaSeq6000 (Illumina, San Diego, CA, USA). The sequencing output was approximately 3 GB per sample.

We compared three different FFPE-RNA isolation procedures on RNA quality, as estimated with the DV₂₀₀, determined by the 4200 TapeStation System (Agilent). We compared the RNeasy FFPE kit (QIAGEN), the ReliaPrep FFPE total RNA miniprep kit (Promega) and the AllPrep DNA/RNA FFPE isolation kit (QIAGEN). For the FFPE-RNA application cohort, total RNA and genomic DNA were isolated from FFPE samples with QIAGEN’s QIAcube classic (cat. no. 9001293; QIAGEN). A DV₂₀₀>15% was deemed sufficient for RNA-seq.