Firescript rt cdna synthesis kit

RNA was extracted from cell pellets using the QIAGEN RNeasy Kit (QIAGEN, 74106). cDNA was synthesized using the FIREScript RT cDNA Synthesis Kit (Solis BioDyne, 06-15-00200); then, qRT-PCR was conducted on a ViiA 7 system (Applied Biosystems) with the HOT FIREPol EvaGreen qPCR master mix (Solis BioDyne, 08-24-00020). Gene cycle thresholds were calculated using triplicate replicates and normalized to housekeeping controls. See table S6 for primer list and sequences.

For quantification of mRNA levels, total RNA was isolated from yeast cultures. To that end, cells equivalent to an OD₆₀₀ of 3 were harvested at indicated time points and resuspended in 1 ml of TRIzol reagent (Thermo Fisher Scientific). Approximately 200 μl of glass beads (500 μm diameter) were added and cells were mechanically lysed in three cycles of 1 min. Subsequent steps were performed as described in the manufacturer’s manual. RNA integrity was visualized by agarose-gel electrophoresis (adapted from Aranda et al., 2012 (link)). Residual DNA was removed with the DNA-free Kit (Ambion) and the RNA concentration was determined with a NanoDrop 1000 Spectrophotometer. Reverse transcription of 3.5 μg isolated RNA was performed with the FIREScript RT cDNA synthesis kit (Solis Biodyne) using the supplied random hexamer primers. After dilution of the resulting cDNA, q-RT-PCR was performed with the 5× HOT FirePol EvaGreen qPCR Supermix (Solis Biodyne). All primers used for q-RT-PCR are listed in Supplementary Table S1. All primers showed an efficiency between 90% and 110%, as assessed with standard curve experiments. To measure gene expression levels, cDNA was utilized in duplicate for q-RT-PCR and the relative gene expression levels were calculated with the comparative C_T method (Livak and Schmittgen, 2001 (link)) with normalization to UBC6 as housekeeping gene.

RNA was extracted from mouse tissue using QIAzol lysis reagent (Qiagen, Hilden, Germany) and subsequently used as template for cDNA synthesis using the FIREScript® RT cDNA synthesis KIT (Solis BioDyne, Tartu, Estonia). cDNA was then used as template in the quantitative-polymerase chain reaction (qPCR) with gene-specific primers and SYBR green dye to determine quantification cycle (Cq) using the Applied Biosystems QuantStudio 6 and 7 Flex Real-Time PCR Systems. Relative DENV expression level was calculated using the ΔΔCq method with cyclophilin A (CPH) cDNA as an internal control. The primer sequences used in the study were: CPH forward 5’-atggtcaaccccaccgtgt-3’, reverse 5’-ttcttgctgtctttggaactttgtc-3’; DENV forward 5’-gactagcggttagaggagacccct-3’, reverse 5’-tcccagcgtcaatatgctgttt-3’.

Total RNA was isolated from venous blood of enrolled individuals using Hybrid-RTM blood RNA extraction Kit (GeneAll Biotechnology Co. Ltd., Seoul, South Korea). The quality and quantity of RNA was appraised using Nanodrop equipment (Thermo Scientific, MA, USA). Subsequently, cDNA was produced using FIREScript RT cDNA Synthesis Kit (Solis BioDyne, Estonia). Relative expressions of lncRNAs were assessed in patients with schizophrenia and controls using RealQ Plus Master Mix Green (AMPLICON, Denmark) in the rotor gene 6000 Real-Time PCR System (Corbett, Australia). B2M gene was used as the normalizer. The sequences of primers and amplicon lengths are shown in Table 1.Table 1

Sequences of primers used in the study.

Primer Name	Sequence	Primer Length	PCR Product Length
MEG3-F	TGGCATAGAGGAGGTGAT	18	111
MEG3-R	GGAGTGCTGTTGGAGAATA	19
SPRY4-IT1-F	AGCCACATAAATTCAGCAGA	20	115
SPRY4-IT1-R	GATGTAGGATTCCTTTCA	18
HOXA-AS2-F	CCCGTAGGAAGAACCGATGA	20	70
HOXA-AS2-R	TTTAGGCCTTCGCAGACAGC	20
Linc-ROR-F	TATAATGAGATACCACCTTA	20	170
Linc-ROR-R	AGGAACTGTCATACCGTTTC	20
UCA1-F	CTTAGGCTGGCAACCATCAGATCC	24	129
UCA1-R	GTGTTGTCCTGCATGCTGGTCTG	23
MALAT1-F	GACGGAGGTTGAGATGAAGC	20	84
MALAT1-R	ATTCGGGGCTCTGTAGTCCT	20
B2M-F	AGATGAGTATGCCTGCCGTG	20	105
B2M-R	GCGGCATCTTCAAACCTCCA	20

Total RNA was extracted from cell pellets using the RNeasy Mini Kit (Qiagen, Cat. No. 74106). RNA quality and concentrations were determined by Nanodrop (Thermo Scientific) spectrophotometric measurements. cDNA synthesis was performed from 300ng of RNA using the FIREScript RT cDNA Synthesis Kit (Solis Biodyne, Cat. No. 06-15-00050) in the Veriti Thermal Cycler (Applied Biosystems). qRT-PCR reactions were set up using the 5x Hot FirePol EvaGreen qRT-PCR SuperMix (Solis Biodyne, Cat. No. 08-36-00001) in QuantStudio 3 Real-Time PCR System (Applied Biosystems). The primers used are listed in Table 1. Gene expression was analyzed using the Comparative C_T (ΔΔ C_T) method after normalization to the housekeeping gene 18s rRNA. All results are presented as fold expression change compared to non-asthmatic healthy controls.

The cDNA was synthesized using a FIREScript RT cDNA synthesis kit (Solis Biodyne, Tartu, Estonia) as previously documented [29 (link),31 (link),32 (link)]. Briefly, the 20 μL reaction was prepared by adding 500 ng of the RNA sample, 100 μM oligo (dT) primers (1 μL), 10 × RT buffer (2 μL), reverse transcriptase (RT; 1 μL), dNTP Mix (0.5 μL), 40 U/μL RNase inhibitor (0.5 μL), and then RNase-free water. The conditions for converting cDNA were as follows: an initial annealing step at 25 °C for 5 min, followed by a reverse transcription step at 45 °C for 15 min, and an RT inactivation step at 85 °C for 5 min. The concentration of cDNA was quantified before further analysis.