Immediately prior to euthanasia, macaques were sedated with ketamine and sodium pentobarbital was administered. Transcardiac perfusion of the entire body with 5 liters of sterile saline was performed to remove blood from the cardiovascular tree and vessels within tissues. Tissues were collected for routine histologic analysis and three sections of each tissue (approximately 3 cm × 1 cm × 0.4 cm) were excised using autoclave-sterilized instruments and placed immediately into 12 ml of RNAlater (Sigma-Aldrich, St. Louis, Missouri). Tissues were allowed to fix in RNAlater at 4°C overnight, then were removed, blotted, cut in sections weighing 20–50 mg, and stored in individual tubes at −80°C. Tissues were homogenized using the TissueLyser II (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and RNA was extracted from each tissue using the Tissue LEV Total RNA Purification Kit (Promega, Madison, WI) on a Maxwell 16 MDx instrument.
Tissue lev total rna purification kit
Manufactured by Promega
Sourced in United States
The Tissue LEV Total RNA Purification Kit is a laboratory equipment product designed for the extraction and purification of total RNA from various tissue samples. It utilizes a simple and efficient protocol to isolate high-quality RNA suitable for downstream applications such as RT-qPCR, Northern blotting, and microarray analysis.
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2 protocols using tissue lev total rna purification kit
1
Tissue Collection for Transcriptomic Analysis
2
Viral Infection Detection in P. vannamei
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The diseased P. vannamei examined in this study were collected from 2016 to 2019 from affected ponds located in the Brazilian northeast region including Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State. The average weights of the diseased juvenile shrimp in the affected ponds were approximately 3.0 gm. Five to six shrimp were collected from each farm and screened by a real-time RT-PCR analysis, H&E histopathology and ISH. RNA isolation was performed using approximately 25 mg of the minced muscle and pleopods of the presumably affected shrimp. The Tissue LEV Total RNA Purification Kit (#AS1220, Promega, Madison, WI, USA) along with an automated DNA/RNA extraction system (Maxwell® MDX Promega, USA) was used following the manufacturer’s recommendations. One-step real-time RT-PCR was carried out following the protocols described by Andrade et al. [16 (link)]. Prior to the real-time assay, extracted RNA was boiled at 100 °C for 3 min to denature dsRNA, then placed on ice and finally subjected to real-time RT-PCR analysis using GoTaq® Probe 1-Step RT-qPCR system (PROMEGA, Madison, WI, USA). Each sample was analyzed in duplicate using a real-time PCR ViiA7 thermocycler (Applied Biosystems, Foster city, CA, USA).
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