Direct zol rna miniprep extraction kit

Using the optic chiasm as a reference, a 1-mm sample corer (Fine Scientific Tools) was used to collect bilateral punches of the SON from hypothalamic coronal slices (Fine Scientific Tools). SON punches were frozen on dry ice, then re-suspended by continuous vortexing for 1 min in 400 µl of QIAzol lysis reagent (Qiagen). Following a 10-min incubation at room temperature, debris was removed by centrifugation at 12,000 × g for 3 min. The supernatant was carefully removed and then mixed with an equal volume of absolute ethanol. Total RNA was extracted with the Direct-zol RNA MiniPrep extraction kit (Zymo Research, Irvine, CA, USA) and then applied to a Zymo-Spin IIC column. RNA was eluted in a volume of 25 µl.

Quantative PCR was performed to verify the expression of embryonic stem cell marker mRNAs (SOX2 and KLF4). A375 cells, in both adherent and spheroid conditions, were harvested and total RNA was extracted using the Direct-zol RNA miniprep extraction kit (Zymo research, Irvine, CA), following the manufacturer’s protocol. RNA concentrations were assessed using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA).
Specific sets of primers for SOX2 and KLF4 cDNAs were designed and synthesized (Sigma-Aldrich, Milano, Italy), according to Miranda-Lorenzo and coworkers^{17 (link)}. Real-time PCR was performed as previously described^{68 (link)}. 18 S levels were used as housekeeping controls. The amplification of gene expression was quantified using the comparative threshold-cycle (DDCt) method.

RNA was extracted from 20 whole bodies, 20 bodies without heads, or 60 heads from 5 day old male flies (unless otherwise indicated) of the indicated genotype, using TRIzol (Life Technologies) and Direct-zol RNA MiniPrep extraction kit (Zymo Research). cDNA was generated using the iSCRIPT cDNA Synthesis kit (Bio-Rad). Quantitative real-time PCR (qPCR)-based quantification was performed for the following genes using the indicated primers:
GBA1a
Forward ACGATGACCAACGCTATTCC
Reverse ATACCAGTGCAGCGATAGCC
dGBA1b
Forward GAACCAGAGCAATCCCTTCA
Reverse TCATCGAGAGTCACGTCCAC
CG31413
Forward CAAGTCCCTTGAAGCCAGAG
Reverse CGAAGGACGAGAGGCAATAC
Rap2L
Forward CCGCTGAAGGTAATGCCTTG
Reverse CGTTTATCCGATCCTTTGCAGA
qPCR was performed using iTaq Universal SYBR Green Supermix (Bio Rad) and a LightCycler 480 (Roche) machine. The delta-delta log₂ method was used to calculate fold change in expression levels. Rap2L was used as the internal control, as the expression of this gene has been reported as the most invariant across different genotypes and ages [66 (link)]. Each experiment was performed at least three times.

The Direct-zol RNA Miniprep Extraction Kit (Zymo Research, Catalog no. R2052) was used to extract the viral RNA from the collected tissues according to the manufacturer’s instructions. Genomic RNA of SARS-CoV-2 was quantified using qRT-PCR with previously described primers and probes.^{7 (link)}