TRIzol Reagent
RNA isolation / purification Tissue - Mouse Cornea
Protocol tips
| Protocol tips |
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| - For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it. |
Publication protocol
Real-time PCR was performed to detect gene expression in corneal epithelium and conjunctival tissues. Total RNA was extracted from mouse corneal epithelia and conjunctival tissues with the use of TRIzol (Invitrogen, Carlsbad, CA) and was reverse-transcribed to cDNA with the use of the ExScript RT Reagent kit (Takara, Dalian, China). The primers used to amplify specific gene products are listed in Table 1. Real-time PCR was performed with a StepOne Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) with the use of a SYBR Premix Ex Taq Kit (Takara) according to the manufacturer's instructions. The amplification program included an initial denaturation step at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds and annealing and extension at 60°C for 30 seconds for 40 cycles. The results of relative quantitative real-time PCR were analyzed by the comparative threshold cycle method and normalized to β-actin expression as an internal control.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
4 years ago
Author:
Paul G. Macon
RNA isolation from tissue
How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?
Discussion
5 years ago
Author:
Aaron Stege
Problem in phase separation after using serum/plasma kit
I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?
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