Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens
A paraffin embedding and sample technology, which is applied in the field of purification of RNA, DNA and protein, and can solve problems such as lack of quantitative technology
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Embodiment 1
[0076] Example 1 Routine RNA isolation steps
[0077] RNA was extracted from paraffin-embedded tissues as follows:
[0078] A. Dewaxing and rehydration of sections:
[0079] (1) Place about 10 μM slices in a 1.5ml plastic centrifuge tube;
[0080] (2) Add 600 μl of xylene, and shake the mixture vigorously for about 10 minutes at room temperature (about 20-25° C.).
[0081] (3) Centrifuge the sample at room temperature for about 7 minutes at the maximum speed of the benchtop centrifuge (about 10-20000 xg).
[0082] (4) Repeat steps 2 and 3 until most of the paraffin is dissolved. Usually two or more passes are required, depending on the amount of paraffin contained in the original sample section.
[0083] (5) Remove the xylene solution by shaking vigorously with a lower alcohol, preferably 100% ethanol (about 600 μl) for about 3 minutes.
[0084] (6) Centrifuge the test tube for 7 minutes as in step (3). The supernatant was gently removed and discarded. The precipitate t...
Embodiment 2
[0096] Embodiment 2 heating time
[0097] This example illustrates the effect of heating time on RNA yield.
[0098] like figure 1 As shown in , heating the chaotropic solution at 95 °C prior to precipitation and reverse transcription significantly increased the detection efficiency of TS and β-actin targets. When the heating step was not included, neither TS nor β-actin could be detected (0 min time point). After 20 minutes at 95°C, both transcripts were detectable; further heating to 60 minutes increased the detection sensitivity of TS by 3-fold and that of β-actin by 4.5-fold. (NRT = no reverse transcriptase control; RT-NRT = total relative gene expression level, ie reverse transcriptase minus no reverse transcriptase).
[0099] figure 2 Shows the RNA expression of β-actin gene in normal (N) and tumor (T) tissues. The samples were heated at 95°C for 0 to 40 minutes. A preferred heating time of about 30 minutes was observed for most samples.
[0100] image 3 It was...
Embodiment 3
[0101] Embodiment 3 heating solution
[0102] This example demonstrates that heating the RNA solution in the presence of a chaotropic agent is important to obtain high yields of RNA. This is a RT-PCR assay using the detection of β-actin gene expression as a measure of the relative amount of RNA isolated from various solutions.
[0103] Clinical samples of esophageal cancer FFPE tissue samples were processed according to the aforementioned method, but the initial pellet obtained after deparaffinization was dissolved or suspended in 4M guanidine isothiocyanate (GITC), 4M guanidine isothiocyanate + 100 μM β-mercaptoethanol (GITC+ BME), 4M guanidine isothiocyanate+20 μM dithiothreitol (GITC+DTT) or Tris-Cl buffer (10 mM, pH 7.5) or Tris-Cl buffer+20 μM DTT (Tris / Cl+DTT). The samples were then heated at 95°C for 30 minutes or not (0 minutes, 95°C). Tris / Cl samples were then treated with 4M guanidine isothiocyanate. RNA levels were determined by detection of β-actin by RT-PCR and...
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