We collected 2 μl of blood from adult zebrafish by making an incision in either the caudal axial vein or the dorsal aorta. The blood was then mixed with 0.5 μl 3.8% sodium citrate in PBS [17 (link)]. Thrombocytes were prepared by the immunoselection method described previously [19 (link)]. Red cells are collected by centrifuging the zebrafish blood at 500 x g for 3 minutes, and the top plasma and white cell layers were removed including a small portion of red cells. Only red cells from the bottom were used. Total RNA from red cells, thrombocytes and embryos were prepared by Absolute RNA kit (Agilent Technologies, Wilmington, DE). For microarray analysis total RNA was isolated from thrombocytes and red cells and was run through columns provided in the RNeasy® Mini Kit (Qiagen Inc., Valencia, CA). These ift122 cDNA templates containing T7 promoter were used to generate sense and antisense ift122 RNA using MEGAshortscript T7 high yield transcription kit (Invitrogen, CA) according to manufacturer’s protocols.
Absolute rna kit
Manufactured by Agilent Technologies
The Absolute RNA kit is a laboratory equipment product designed for the isolation and purification of RNA from various biological samples. It provides a standardized and efficient method for extracting high-quality RNA for downstream applications.
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Lab products found in correlation
2 protocols using absolute rna kit
1
Zebrafish Blood Collection and RNA Extraction
2
Quantitative Gene Expression Analysis
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Total RNA was isolated using the Absolute RNA Kit from Stratagene (Agilent Technologies) and cDNAs were synthesized using SuperSript III First strand cDNA synthesis system for RT-PCR (Invitrogen) according to manufacturer’s instructions. qPCR was performed using the ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems) with a SYBR Green PCR Master Mix kit (Applied Biosystems) and a standard temperature protocol. The results are expressed as relative quantities and calculated using the 2-ΔΔCT method. Three separate qPCR experiments were performed. Actin was used as a control gene for normalization. Primers were purchased from Qiagen (QuantiTect primer assay, IL-33, IL-8, IL-6, SELE) or Sigma Genosys (Actin, NFKB2, ICAM1, VCAM1, ST2).
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