【求助】如何提取线粒体总RNA？谢谢各位。
丁香园论坛
4904
1、如何从组织/细胞中提取线粒体总RNA？请各位大牛指点，谢谢。
2.目的基因编码的蛋白质A在线粒体中有表达，此蛋白质A是一种核编码的蛋白质；请问能否从线粒体中提取到A蛋白的mRNA?如何提取？
谢谢各位指点。
2.目的基因编码的蛋白质A在线粒体中有表达，此蛋白质A是一种核编码的蛋白质；请问能否从线粒体中提取到A蛋白的mRNA?如何提取？
谢谢各位指点。
应该只能梯度离心分离细胞器然后分离MRNA了吧，
如果蛋白是表达在线粒体里应该能在线粒体中提取到其MRNA毕竟要运送到核糖体进行翻译的，
不过量就难以判断了，可以试试！
如果蛋白是表达在线粒体里应该能在线粒体中提取到其MRNA毕竟要运送到核糖体进行翻译的，
不过量就难以判断了，可以试试！
仅供参考：
Mitochondrial Protein Extraction:
a) Collect cells by centrifugation at approximately 370g for 10 minutes. Decant supernatant and re-suspend cells in 10 packed cell volumes of NKM buffer (1 mM Tris-HCl pH 7.4; 0.13 mM NaCl; 5 mM KCl; 7.5 mM MgCl2).
b) Pellet cells and decant supernatant, repeat this washing step 2 times. Resuspend cells in 6 packed cell volumes of homogenization buffer (10 mM Tris-HCl pH 6.7; 10 mM KCl; 0.15 mM MgCl2; 1 mM PMSF; 1 mM DTT; add PMSF and DTT immediately before use).
c) Transfer cells to a glass homogenizer and incubate for 10 minutes on ice. Using a tight pestle, homogenize the cells. Check under microscope for cell breakage ( the optimum is around 60%).
d) Pour homogenate into a conical centrifuge tube containing 1 packed cell volume of 2 M sucrose solution and mix gently. Pellet unbroken cells, nuclei and large debris at 1200 g for 5 minutes and transfer the supernatant to other tube. This treatment is repeated twice, transferring the supernatant to a new tube each time, discarding the pellet.
e) Pellet the mitochondria by centrifugation at 7000 g for 10 minutes. Resuspend the mitochondria pellet in 3 packed cell volumes of suspension buffer (10 MM Tris-HCl pH 6.7; 0.15 mM MgCl2; 0.25 mM sucrose; 1 mM PMSF; 1 mM DTT). Spin at 9500 g for 5 minutes to re-pellet mitochondria.
f) At this point you can add 1x protein loading buffer and run on a gel if a whole mitochondrial protein extract is needed, further purify the mitochondria on a sucrose gradient if a very pure extract is needed. To purify the soluble (S-100) mitochondrial fraction see protocol below.
Soluble (S-100) Mitochondrial Protein Extraction:
a) Ressupend mitochondria in 1/3 the packed cell volume lysis buffer (25 mM HEPES-KOH pH 7.6; 5 mM MgCl2; 0.5 mM EDTA; 10% Glycerol; 1 mM DTT; 1 mM PMSF). Put the suspension into a glass homogenizer and homogenize with a tight pestle. Add 0.5% Tween 20 and 0.5 M KCl.
b) Incubate the mixture on ice for 5 minutes. The homogenization is repeated 10 times.
c) Spin the final mitochondrial lysate at 100,000 g in a ultracentrifuge at 4?C for 60 minutes.
d) Carefully collect the clear supernatant, avoiding the fluffy layer over the pellet, to yield the final S-100 fraction.
e) Freeze in aliquots, in liquid nitrogen and store at -80?C.
(from http://www.frilabo.pt/fcms/index.php)
Mitochondrial Protein Extraction:
a) Collect cells by centrifugation at approximately 370g for 10 minutes. Decant supernatant and re-suspend cells in 10 packed cell volumes of NKM buffer (1 mM Tris-HCl pH 7.4; 0.13 mM NaCl; 5 mM KCl; 7.5 mM MgCl2).
b) Pellet cells and decant supernatant, repeat this washing step 2 times. Resuspend cells in 6 packed cell volumes of homogenization buffer (10 mM Tris-HCl pH 6.7; 10 mM KCl; 0.15 mM MgCl2; 1 mM PMSF; 1 mM DTT; add PMSF and DTT immediately before use).
c) Transfer cells to a glass homogenizer and incubate for 10 minutes on ice. Using a tight pestle, homogenize the cells. Check under microscope for cell breakage ( the optimum is around 60%).
d) Pour homogenate into a conical centrifuge tube containing 1 packed cell volume of 2 M sucrose solution and mix gently. Pellet unbroken cells, nuclei and large debris at 1200 g for 5 minutes and transfer the supernatant to other tube. This treatment is repeated twice, transferring the supernatant to a new tube each time, discarding the pellet.
e) Pellet the mitochondria by centrifugation at 7000 g for 10 minutes. Resuspend the mitochondria pellet in 3 packed cell volumes of suspension buffer (10 MM Tris-HCl pH 6.7; 0.15 mM MgCl2; 0.25 mM sucrose; 1 mM PMSF; 1 mM DTT). Spin at 9500 g for 5 minutes to re-pellet mitochondria.
f) At this point you can add 1x protein loading buffer and run on a gel if a whole mitochondrial protein extract is needed, further purify the mitochondria on a sucrose gradient if a very pure extract is needed. To purify the soluble (S-100) mitochondrial fraction see protocol below.
Soluble (S-100) Mitochondrial Protein Extraction:
a) Ressupend mitochondria in 1/3 the packed cell volume lysis buffer (25 mM HEPES-KOH pH 7.6; 5 mM MgCl2; 0.5 mM EDTA; 10% Glycerol; 1 mM DTT; 1 mM PMSF). Put the suspension into a glass homogenizer and homogenize with a tight pestle. Add 0.5% Tween 20 and 0.5 M KCl.
b) Incubate the mixture on ice for 5 minutes. The homogenization is repeated 10 times.
c) Spin the final mitochondrial lysate at 100,000 g in a ultracentrifuge at 4?C for 60 minutes.
d) Carefully collect the clear supernatant, avoiding the fluffy layer over the pellet, to yield the final S-100 fraction.
e) Freeze in aliquots, in liquid nitrogen and store at -80?C.
(from http://www.frilabo.pt/fcms/index.php)
谢谢，线粒体蛋白提取目前来说方法比较成熟。但是就是mRNA的提取很少见到文献报道。
线粒体的mRNA提取果然是比较少。我找到了两篇文献中有提到，希望有所帮助。
先收下两篇文献研究一下
还在纠结怎么弄呢，郁闷ing
还在纠结怎么弄呢，郁闷ing
提好线粒体，再提RNA吧。有难度，量估计少。
有难度，折腾怎么把线粒体提出来，担心耗时太长，RNA被降解了。
降解有这种可能，但提取的线粒体可以进行各种生物化学功能分析，说明线粒体是有活性的，所以，其RNA的降解也许是有限的！
现在也在琢磨着怎样提！
现在也在琢磨着怎样提！
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