Total RNA from conjunctiva or the corneal epithelium was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) following the manufacturer’s protocol. Conjunctiva was surgically excised. One sample equaled the tissue pooled from both eyes of each animal. After isolation, the concentration of RNA was measured and cDNA was synthesized using the Ready-To-Go™ You-Prime First-Strand kit (GE Healthcare) as previously described15 (link). Real time PCR was performed using specific Taqman probes for IL-17A (Il17a, Mm0043918_m1), IFN-γ (Ifn-γ, Mm00801778_m1), IL-13 (Il13, Mm99999190_m1), Foxa2 (Foxa2, Mm00839704_mH), Integrin α2 (Itga2 or DX5, Mm00434371_m1) (Taqman Universal PCR Master Mix AmpErase UNG) in a commercial thermocycling system (StepOnePlus™ Real-Time PCR System, Applied Biosystems), according to the manufacturer’s recommendations. The beta-2 microglobulin (β2 m) (B2m) (Mm00437762_m1) gene was used as an endogenous reference for each reaction. The results of quantitative PCR were analyzed by the comparative Ct method in which the target of change = 2−ΔΔCt and were normalized by the Ct value of β2 m and the mean Ct of relative mRNA level in the untreated naïve group. Gene expression of 3–4 mice per group per time point in two independent experiments with a total of 6–8 mice was determined.
Ready to go you prime first strand kit
Manufactured by GE Healthcare
Sourced in United States
The Ready-To-Go™ You-Prime First-Strand kit is a lab equipment product designed for the synthesis of first-strand cDNA from total RNA samples. The kit provides all the necessary reagents for the reverse transcription reaction, enabling researchers to efficiently generate first-strand cDNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Rneasy plus mini rna isolation kit, by Qiagen (4 mentions)
Rneasy plus micro rna isolation kit, by Qiagen (3 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mimi kit, by Qiagen (1 mentions)
No amperase ung, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix 2x, by Thermo Fisher Scientific (1 mentions)
8 protocols using ready to go you prime first strand kit
1
Quantitative RT-PCR Analysis of Conjunctival Gene Expression
2
Quantitative Analysis of STRA6 and RBP-4
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Following euthanasia, the corneal epithelium was scraped, and total RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. The cDNA was synthesized using the Ready-To-Go−You-Prime-First-Strand kit (GE Healthcare, Pittsburgh, PA, USA). Quantitative real-time PCR was performed with specific Taqman probes (Life Technologies, Grand Island, NY, USA) for STRA6 (Mm00486457_m1) and RBP-4 (Mm00803264_g1). The HPRT-1 gene was used as an endogenous reference for each reaction. The results of real-time PCR were analyzed by the comparative CT method.
3
Extraction and Analysis of Lacrimal Gland RNA
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Total RNA from lacrimal glands was extracted using a QIAGEN RNeasy Plus Mini RNA isolation kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA concentration was measured and 1 ug of total RNA was used to synthesize cDNA (Ready-To-Go™ You-Prime First-Strand kit, GE Healthcare, Chicago, IL, USA). Real-time PCR was performed using specific MGB Taqman probes for IFN-γ (Ifng, Mm01168134), IL-12 (il12a, Mm00434165), major histocompatibility complex class II (CIITA, Mm00482914), TNF-α (TNF, Mm00443258), IL-1β (Il1b, Mm00434228), Cathepsin S (CTSS, Mm 01255859), Unc-51 like autophagy activating kinase (ULK)1 (ULK1, Mm00437238), ULK2 (ULK2, Mm03048846), Beclin1 (BECN1, Mm01265461), Autophagy related (ATG)5 (ATG5, Mm01187303), Microtubule-associated protein 1B-light chain (LC3)b (MAP1LC3B, Mm00782868) using a Taqman Universal PCR system (StepOnePlus™ Real-Time PCR System, Applied Biosystems, Bedford, MA, USA). The hypoxanthine phosphoribosyltransferase 1 (HPRT1, Mm00446968) gene was used as a housekeeping gene for each reaction. Ct values were then normalized by HPRT values [71 (link)].
4
Quantitative Analysis of Lacrimal Gland mRNA
5
Corneal Gene Expression Analysis
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Following euthanasia, the cornea/conjunctiva was excised and total RNA was extracted using an RNeasy® Plus Mimi Kit (Cat No. 74134, QIAGEN GmbH, Hilden, Germany) according to manufacturer's instruction. The RNA concentration was measured, and cDNA was synthesized using the Ready-To-Go-You-Prime-First-Strand kit (GE Healthcare). Quantitative real-time PCR was performed with specific probes Murine MGB probes, Cxcl16 (Mm00801778), Sprr2a (Mm00845122_s1), Sprr2f (Mm00448855_s1), Sprr2g (Mm01326062_m1), Vegfa (Mm00437304), Vegfb (Mm00442102), Vegfc (Mm00437310), Tnf (Mm00443260), Fgf7 (Mm00433291), Mmp9 (Mm00442991), and hypoxanthine phosphoribosyltransferase (Hprt1, Mm00446968). The Hprt-1 gene was used as an endogenous reference for each reaction. The results of real-time PCR were analyzed by the comparative CT method. The CT values of Pinkie were compared to that of B6.
6
Gene Expression in Lacrimal Glands
7
Transcriptome Analysis of Lacrimal Glands
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We extracted RNA from excised extraorbital lacrimal glands (male, number of glands: YY = 10, AA = 10, YA-Y = 10, YA-A = 10; female, number of glands: YY = 10, AA = 6, YA-Y = 10, YA-A = 10) according to the manufacturer’s protocol, using a QIAGEN RNeasy Plus Mini RNA isolation kit (Qiagen, Hilden, Germany). To generate cDNA, we measured the concentration of RNA, calculated 1 µg of RNA for use, and then used the Ready-To-Go™ You-Prime First-Strand kit (GE Healthcare, Chicago, IL, USA). All real-time PCR used minor groove-binding Taqman probes, which were IFN-γ (Ifng, Mm01168134), major histocompatibility complex class II (Ciita, Mm00482914), TNF-α (Tnf, Mm00443258), IL-1β (Il1b, Mm00434228), Cathepsin S (Ctss, Mm00457902), CXCL13 (Cxcl13, Mm00444533), CXCL9 (Cxcl9, Mm00434946), and CD19 (Cd19, Mm00515420). Real-time PCR reactions used TaqMan™ Fast Universal PCR Master Mix (2X) and no AmpErase™ UNG (Thermo Fisher Scientific) and were all carried out on a Taqman Universal PCR system (StepOnePlus™ Real-Time PCR System, Applied Biosystems, Bedford, MA, USA). A housekeeping gene, hypoxanthine phosphoribosyltransferase 1 (HPRT1, Mm00446968), was used to normalize all Ct values.
8
Lacrimal Gland RNA Extraction and Gene Expression Analysis
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