High capacity reverse transcription cdna synthesis kit

RNA samples were prepared using TRIzol (Invitrogen) according to the manufacturer’s recommendations. Spectrophotometry was employed for RNA quantification. For each sample, 2 μg of RNA was employed for the synthesis of complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Synthesis kit (Applied Biosystems) according to the manufacturer’s recommendations. Real-time PCR reactions were run using the TaqMan system (Applied Biosystems). Primers used were GLAST (Mm00600697_m1); GLUT1 (Mm00441480_m1); FGF10 (Mm00433275_m1); IGFBP2 (Mm00805581); DCX (Mm00438400_m1); GFAP (Mm01253033_m1), AGRP (Mm00475829_g1); NPY (Mm00445771_m1); NEUROD1 (Mm01946604_s1); SOX2 (Mm03053810_s1); HCRT (Mm01964030_s1); POMC (Mm00435874_m1); GPR40 (Mm00809442_s1) SOX11 (Mm01281943_s1); Nestin (Mm00450205_m1); RAX (Mm01258704_m1); and BDNF (Mm01334043_m1). Analyses were run using 4 μL (10 ng/μL) cDNA, 0.625 μL primer/probe solution, 1.625 μL H₂O, and 6.25 μL 2X TaqMan Universal MasterMix. GAPDH (Mm99999915_g1) was employed as a reference gene. Gene expression was obtained using the SDS System 7,500 software (Applied Biosystems).

Total RNA was isolated using TRIzol^® reagent (Ambion, #15596018). A total of 500 ng of RNA was reverse transcribed to cDNA using the High-capacity cDNA synthesis reverse transcription kit (Applied Biosystems, #4368813), as per the manufacturer’s instructions. The cDNA was used for measurement of Ceacam1, viral M gene, and viral N gene expression in MHV-infected Cas9-expressing iBMDMs with or without Ceacam1 depletion by gRNA treatment (described above). mRNA levels were quantified in real time using Sybr^® Green (Applied Biosystems, #4367659) and the Quant Studio™ 7 Flex Real-Time PCR System (Applied Biosystems). The expression levels were normalized to Rpl32 in the case of the viral genes and Actb for the host cellular Ceacam1 gene expression and presented as fold change. Forward and reverse primer sequences used in the study are as follows: M gene, Forward: 5-GGAACTTCTCGTTGGGCATTATACT-3, Reverse: 5-ACCACAAGATTATCATTTTCACAACATA-3; N gene, Forward: 5-CAGATCCTTGATGATGGC GTAGT-3, Reverse: 5- AGAGTGTCCTATCCCGACTTTCTC-3; Rpl32, Forward: 5-AAGCGAAA CTGGCGGAAAC-3, Reverse: 5-TAACCGATGTTGGGCATCAG-3; Ceacam1, Forward: 5-ATTT CACGGGGCAAGCATACA-3, Reverse: 5-GTCACCCTCCACGGGATTG-3; and Actb, Forward: 5-CAGCTTCTTTGCAGCTCCTT-3, Reverse: 5-CACGATGGAGGGGAATACAG-3.

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate RNA according to manufacturer’s instructions. The NanoDrop-2000 spectrophotometer (ThermoScientific, Waltham, MA, USA) was used to determine total RNA yield. cDNA synthesis was done in a thermocycler (AB Applied Biosystems, Foster City, CA, USA), after mixing 1 μg RNA and a master mix prepared with the high-capacity cDNA synthesis reverse transcription kit (AB Applied Biosystems). SYBR® Green PCR Master Mix (Applied Biosystems) was used to perform a Quantitative polymerase chain reaction (PCR) looking at the following genes of interest: Il-6, Il-10, Tgf-β1, Tgf-β3, Lcn-2, iNos, Arg, and Tnf-α.

Tissue samples (5-10 mg) were lysed using Qiagen Tissue lyser II and total RNAs were isolated using mirVana kit (ThermoFisherScientific, USA) after DNase treatment. The purity and quantity of RNA were measured using NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, USA) and integrity was checked by determining RNA integrity number (RIN) using an Agilent Bioanalyzer (2100). The cDNA was synthesized from total RNA (1 μg) using a High-capacity cDNA synthesis reverse transcription kit (Applied Biosystems, USA) as per the manufacturer’s instruction manual.

Total RNA was isolated by Trizol reagent (Sigma). The isolated RNA was quantified using NanoDrop UV Spectrophotometer. Two microgram of total cellular RNA was reverse transcribed using High Capacity cDNA Synthesis Reverse Transcription Kit (Applied Biosystems, MA, USA). Real-time (RT)-PCR was performed in Applied Biosystems StepOne Plus using SYBR green, and the data were normalized using β-actin as endogenous control. The fold change of the target genes were analyzed using the comparative CT method.

Total RNA was obtained from cell samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Detection and quality control of the RNAs were performed using a NanoDrop‐2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using a thermocycler (Applied Biosystems, Foster City, CA, USA) after mixing 20 μL of RNA and a master mix prepared with the High‐Capacity cDNA Synthesis Reverse Transcription kit (Applied Biosystems). RT‐PCR was conducted using a SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. The PCR cycling conditions were 95°C for 10 minutes followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. All gene expression data were normalized against the values for glyceraldehyde 3‐phosphate dehydrogenase (GAPDH).