Total RNA from the retina was isolated using Nucleospin RNA columns (Clontech, CA, USA). Isolated RNA was reverse transcribed using RNA to cDNA EcoDry (Clontech) following the manufacturer's instructions. Quantitative PCR (qPCR) was performed using KAPA SYBR FAST Universal qPCR kit (KAPA Biosystems, KK4602, Roche Diagnostics, IN, USA), with primers ordered from Integrated DNA Technologies (IDT, CA, USA), and a Rotor Gene Q thermocycler (Qiagen). The qPCR was performed with cDNAs synthesized from 1 μg of the total RNA of each group as a template and specific primers (sTable 2 ).
Nucleospin rna column
Manufactured by Takara Bio
Sourced in Japan
The Nucleospin RNA columns are a tool used for the isolation and purification of total RNA from various biological samples. The columns are designed to efficiently capture and retain RNA, allowing for its separation from other cellular components.
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Lab products found in correlation
5 protocols using nucleospin rna column
1
Retinal Total RNA Isolation and qPCR
2
Total RNA Extraction and qPCR Analysis
3
Quantifying Gene Expression by qPCR
4
Transcriptional Profiling of Metabolic Genes
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The transcriptional expression of maeB, ppsA, ppc, pckA, pgi, zwf, gltA, and arcA in each strain was quantified using real-time PCR. Total RNA was isolated from individual cultures using a NucleoSpin RNA column (Takara Bio, Shiga, Japan) according to the manufacturer's protocol. Reverse transcription reactions and quantitative real-time PCR was performed using an Mx3005P Real-Time QPCR System (Agilent Technologies, Santa Clara, CA, USA) with RNA-direct SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan). The primer pairs are listed in Supplementary Data 7 . The normalized transcriptional level of each mRNA was calculated by the relative quantification method using the mdoG gene (coding glucan biosynthesis protein G) as the housekeeping gene61 (link).
5
Transcriptional Analysis of Aromatic Amino Acid and Lactate Dehydrogenase Genes
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The transcriptional expression of aroB, aroG, and ldh in SA-2, SA-5, and SA-7 was quantified using real-time qPCR. Total RNA was isolated after 24 h of cultivation in CGXIIY medium using a NucleoSpin RNA column (Takara Bio, Shiga, Japan) according to the manufacturer’s protocol.
Quantitative real-time qPCR was performed using a LightCycler® 96 System (Roche Molecular Systems, Inc., CA, United States) with RNA-directTM Real-time PCR Master Mix (TOYOBO). The primer pairs used are listed inSupplementary Table 1 . The normalized transcriptional level of each mRNA was calculated using the relative quantification method using the cg3177 (Ncgl2772) gene as the housekeeping gene.
Quantitative real-time qPCR was performed using a LightCycler® 96 System (Roche Molecular Systems, Inc., CA, United States) with RNA-directTM Real-time PCR Master Mix (TOYOBO). The primer pairs used are listed in
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