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PicoPure

Manufactured by Arcturus

The PicoPure is a compact and versatile laboratory instrument designed for precise and accurate measurements. It features a high-resolution sensor and advanced data processing capabilities to deliver reliable results. The core function of the PicoPure is to provide accurate and reliable measurements for a variety of laboratory applications.

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4 protocols using PicoPure

RNA was extracted from the cells following the TRIzol LS protocol guidelines, until the isoproponal precipitation step, then RNA was re-suspended in extraction buffer of a RNA isolation kit (PicoPure, Arcturus), and isolation continued according to the manufacturer’s guideline. This two-step purification protocol helps obtaining RNA of high quality when starting with samples of large volumes, and resulted in a yield of around 8−24 ng per replica with RIN ≥ 8, as measured by a Bioanalyzer (Agilent). Specificity of the RNA was determined by using ~7% of purified RNA for first strand cDNA synthesis with SuperScript III kit (Invitrogen), followed by qPCR (LightCycler 480, Roche) analysis, using lmn-1 as a control to standardized the samples, and hlh-17 to measure differential expression between samples derived from the CEPsh glia to the control. All subsequent steps were performed by the Rockefeller University Genomics Resource Center. Briefly, mRNA amplification and cDNA preparation were performed using the SMARTer mRNA amplification kit (Clontech). Labeled samples were sequenced using an Illumina HiSeq 2000 sequencer using either 50- or 100-base read protocols.
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RNA was extracted from the sorted cells following the TRIzol LS protocol guidelines, until the isopropanol precipitation step, then RNA was re-suspended in extraction buffer of an RNA isolation kit (PicoPure, Arcturus) and isolation continued according to the manufacturer’s guideline. This two-step purification protocol helps obtain RNA of high quality when starting with samples of large volumes and resulted in a yield of around 10–80 ng per replica with RNA integrity number (RIN) ≥8, as measured by a Bioanalyzer (Agilent). All subsequent steps were performed by the Rockefeller University Genomics Resource Center. Briefly, mRNA amplification and cDNA preparation were performed using the SMARTer mRNA amplification kit (Takara #634940). Labeled samples were sequenced using an Illumina NextSeq 500 sequencer using 75 base pair single read protocols.
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Eggs were dissected from female bodies by mechanical isolation of tissues in ice cold PBS. Extracted oocytes were counted and moved immediately to Arcturus PicoPure (Ref: 12,204–01) RNA extraction buffer. The entire clutch of oocytes for a single-female was combined as one pooled sample. Seven PP, and seven LL clutches were used. A total of ten F1 oocyte pools were sampled, five from PL females, and five from LP females. A clutch of pooled oocytes is roughly the same amount of physical material regardless of maternal type, and the total RNA yield of clutches was similar in preliminary tests of the RNA extraction protocol as quantified by Qbit chemistry. Total input mRNA will not affect results of statistical tests in subsequent analyses due to established data normalization pipelines. Libraries were constructed with the NEB UltraII Stranded RNA library prep kit for Illumina. Libraries were sequenced on two lanes of 150 bp on the Illumina NovaSeq resulting in 80 million reads/library.
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Mutation analysis of the EGFR gene was conducted as described previously [35 (link)]. In brief, DNA was extracted from paraffin tissue samples using a DNA extraction kit (Arcturus PicoPure) and the tyrosine kinase domain of EGFR was amplified by polymerase chain reaction. The amplicons were purified and sequenced by an automatic ABI PRISM DNA analyzer with technical support from TR6 pharmacogenomic lab, MOST Taiwan [35 (link)]. Two types of EGFR mutations were evaluated with direct sequencing, namely, the deletion in exon 19 and the L858 point mutation in exon 21.
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