Prism 7500 sequence detection system

Following sacrifice, right lungs were immediately homogenized in 2 mL of Trizol reagent and placed in dry ice. The homogenates were stored at − 85 °C until RNA isolation was completed following the manufacturer’s (Sigma, Oakville, Ontario, Canada) instructions. These experiments were performed as described previously (Haston et al. 2005 (link); Honeyman et al. 2013 (link); Lemay and Haston 2005 (link)). Briefly, 4–5 µg of total RNA from the right mouse lung was reverse transcribed with oligo(dT)₁₂₋₁₈ Primer using Superscript™ II RNase H Reverse Transcriptase (Invitrogen, Carlsbad, CA) to make cDNA. Quantitative RT-PCR assays were completed using the Applied Biosystems International Prism 7500 Sequence Detection System and TaqMan™ assays on demand and with the reference gene Ataxin 10 (Atxn10, Assay Mm00450332_m1).

Total RNA from the cervical spinal cord was extracted and then transcribed into cDNA as we previously described.^{49 (link)} Real-time PCR (RT-PCR) was performed with SYBR® and the following oligonucleotide primers mIL-1β, forward 5ʹ-TGG TGT GTG ACG TTC CCA TT-3ʹ, reverse 5ʹ-TCC ATT GAG GTG GAG AGC TTT C-3ʹ; mTNF-α, forward 5ʹ-AGA GGC ACT CCC CCA AAA GA-3ʹ, reverse 5ʹ-CGA TCA CCC CGA AGT TCA GT-3ʹ; mIL-4, forward, 5ʹ-CCACGGATGCGACAAAAATC-3ʹ, reverse 5ʹ-GACGTTTGGCACATCCATCTC-3ʹ; mIL-10, forward 5ʹ-TGA ATT CCC TGG GTG AGA AGC TGA-3ʹ, reverse 5ʹ-TGG CCT TGT AGA CAC CTT GGT CTT-3ʹ; mIL-6, forward 5ʹ-TCC AGA AAC CGC TAT GAA GTT C-3ʹ, reverse 5ʹ-CAC CAG CAT CAG TCC CAA GA-3ʹ; and mRps29 forward 5ʹ-GCC GCG TCT GCT CCA A-3ʹ, reverse 5ʹ-ACA TGT TCA GCC CGT ATT TGC-3ʹ. PCR amplification were initiated incubating at 50°C for 2 min and 95°C for 10 min, and amplification was performed over 40 cycles at 95°C for 15 s and 60°C for 1 min. The cDNA samples were analyzed in triplicate on an Applied Biosystems PRISM 7500 Sequence detection system, normalizing the mRNA expression to the Rps29 gene in each sample and quantifying gene expression by the 2^−ΔΔCt method. The results are expressed relative to the Sham mice for each time point.

The rats were euthanized with an overdose of sodium pentobarbital (200 mg/kg, i.p.), and the brain tissues were collected and cut into 500 μm coronal sections. PVN tissues were dissected by punch-out technique as previously described [20 (link)], and pieces of cerebral cortex from the same section were also obtained. Total RNA from PVN and cerebral cortex was isolated in accordance with the procedure of the RNeasy kit (Qiagen, Valencia, CA, United States). Following reverse transcription of total RNA, the mRNA quantifications for NPY, NPY Y1R, and NPY Y5R were performed through TaqMan real-time PCR method as previously described [21 (link)]. TaqMan PCR probes for rat NPY(Rn01410145_m1), NPY Y1R (Rn02769337_s1), NPY Y5R (Rn02089867_s1), and 18S rRNA (Rn03928990_g1) were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). Applied Biosystems PRISM 7500 sequence detection system was used to detect the PCR reactions. Quantitative analysis of target mRNA expression was performed with a comparative cycle of threshold (CT) fluorescence technology. The expression level of the target genes was normalized by the expression of the 18S rRNA. The arbitrary units of target mRNA were expressed as the ratio of the target mRNA concentration to the 18S rRNA of the same sample.

Centriole-related genes (i.e., cep57, cetn1, rootletin, and nek2) involved in spermatozoa development were selected for verification and comparison in 2nRCC and 3nDTCC using qPCR. Total RNA was extracted from the remaining testicular tissue and reverse-transcribed into first-strand cDNA using reverse transcriptase (Invitrogen). Real-time PCR was performed using the Prism 7,500 Sequence Detection System (Applied Biosystems, United States) with a PowerUpTM SYBRTM Green Master Mix (Applied Biosystems, United States). Real-time qPCR was performed in triplicates.
The reaction mixture (10 µL) consisted of the following components: 2.5 µL of cDNA (1:4 dilution), 5 µL of PowerUpTM SYBRTM Green Master Mix, 0.5 µL of the specific forward primers, 0.5 µL of the reverse primers, and 1.5 µL of water. The amplification conditions were 50°C for 5 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 45 s. The average threshold cycle (Ct) was calculated for each sample using the 2-ΔΔCt method and normalized according to β-actin. Finally, melting curve analysis was performed to validate the specific amplification of the expected products.

The Altona Diagnostics RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Hamburg, Germany) was the primary assay used for molecular diagnosis. A subset of SARS-CoV-2 positives were identified using the Cepheid Xpert Xpress SARS-CoV-2 GeneXpert platform per manufacturer instructions. All samples were processed within 24 hours of collection.
The RealStar® SARS-CoV-2 RT-PCR Kit 1.0 total reaction volume was 30μL (10μL extracted sample and 20μL Master Mix). The kit contains two premade master mixes, A and B, which contain PCR buffer, magnesium salt, primers and probes, reverse transcriptase, and DNA polymerase. The detectors used are Cy5 (SARS-CoV-2; S gene), FAM (B-βCoV; E gene) and JOE (Internal Control). C_T values for the S gene target (Cy5) are reported in Table S2. Taqman RT- PCR was performed using the Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) at the following cycling conditions: 1 cycle at 55.0°C for 20 minutes, 1 cycle at 95.0 °C for 2 minutes and 45 cycles at 95.0°C for 15 seconds, 55.0°C for 45 seconds then 72.0 °C for 15 seconds. Validation of this assay was performed as described in^{22 (link)}.
Testing on the Cepheid Xpert Xpress SARS-CoV-2 GeneXpert platform was performed in accordance with manufacturers instructions³⁴ .

To verify the reliability of the RNA-seq results, eight DEGs (CDKL1, CKB, AHCY, ARHGEF3, TGFβ, SCP1, WNT11 and CYP27A) involved in the development of ovarian tissues were selected for validation using quantitative real-time PCR (qPCR) on a Prism 7500 Sequence Detection System (Applied Biosystems, United States) with a miScript SYBR Green PCR Kit (Qiagen, Germany). The reaction mixture (10 μL) comprised 2.5 μL cDNA (1:3 dilution), 5 μL SYBR Premix Ex TaqTMII (TaKaRa), 0.5 μL specific forward primer, 0.5 μL reversal primer, and 1.5 μL water. Real-time PCR was performed on biological replicates in triplicate. The amplification conditions were as follows: (1) 50°C for 5 min, (2) 95°C for 10 min and (3) 40 cycles at 95°C for 15 s, followed by 60°C for 45 s. The average threshold cycle (Ct) was calculated for 4nRR fish and RCC using the 2^-ΔΔCt method (Pfaffl, 2001 (link)) and normalized to that of β-actin. Finally, a melting curve analysis was completed to validate the specific generation of the expected products.

Total RNA was extracted from the cells and tissues, and the mRNA levels of the target genes were assayed by qRT- PCR. Briefly, total RNA was extracted from the primary cultured GECs and 5/6 nephrectomized kidney tissues using the RNeasy kit (Qiagen GmbH), and 500 ng of total RNA was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega). qRT- PCR was performed using either Assay-on-Demand TaqMan probes or the SYBR Green method and primers for PROS1, LGALS1, NFKB1, P53, FN1, and GAPDH (Applied Biosystems) and analyzed using the Applied Biosystems PRISM 7500 sequence detection system. Data were normalized to GAPDH and presented as fold increase compared with RNA isolated from the control group using the 2^−ΔΔCT method (28 (link)). All experiments were performed in triplicate. PCR primers used for qRT-PCR are listed in supplemental Table S1.