TRIzol Reagent
RNA isolation / purification Tissue - Human 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
Total RNA was extracted using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was generated from 500 ng of RNA with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Gene expression was measured by real-time PCR (Viia7, Thermo Fisher Scientific) using PCR primers (Oligo, Warsaw, Poland) and probes (Roche Universal Probe Library) designed with the ProbeFinder software, version 2.50 (Roche). The analyzed coding region of TCF4 is present in both TCF4-A and TCF4-B isoforms. It is located in the amino-terminal part of the protein, close to the activation domain 2 (AD2). For TCF4 quantitative PCR (qPCR), the primer pair 5′-GCACTTTCCCTAGCTCCTTCT-3′ and 5′-GCATAGCCAGGCTGATTCAT-3′ and probe 25, for RPL13A (60S ribosomal protein L13a) qPCR the primer pair 5′-CTGGTGCTTGATGGTCGAG-3′ and 5′-GTTGATGCCTTCACAGCGTA-3′ and probe 77 were used. Expression values for TCF4 were calculated using the modified double delta Ct (ΔΔCt) method and absolute quantification and normalized to RPL13AFull 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
4 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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Manufacturer protocol
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