Hello to everybody, kto-nibud` vydelial RNA iz krovi? Kak vy eto delali, i kakoi u vas byl vyhod? Bolee konkretno, mojno li iz 100-300 mkl krovi vydelit` 1-2 mkg RNA (dlia RT-PCR)? A. [ 11-12-2002: Сообщение отредактировано: a26 ]
Tom1, 11.12.2002 19:30
уже был такои топик посмотри в архиве форума. [Текст переведён с транслита]
a26, 11.12.2002 20:06
V archive foruma, deistvitel`no, mnogo topikov o krovi, no obsujdeniia nujnogo mne voprosa ia ne nashla (mojet, ploho iskala?). Menia interesuet, est` li u kogo-nibud` proverennaia metodika vydeleniia RNA iz krovi s vyhodom minimum 10 mkg/mL? [ 11-12-2002: Сообщение отредактировано: a26 ]
EKalina, 11.12.2002 21:31
Очень сомневаюсь, что у кого-нибудь окажется такая методика. На страничке выделения РНК из тканей мышки есть таблица с выходами РНК на мг ткани. Больше 11 мкг на 1 мг не видно. А с кровью хорошо сначала от эритроцитов отделаться. И выход лейкоцитов очень низкий. Впрочем, хотелось бы ошибаться.
a26, 11.12.2002 22:39
Vot. Ia v opisanii kita iz "Promega" prochitala, chto vyhod okolo 1 mkg RNA/1 mL krovi. Po moim predstavleniiam eto malo, doljno byt` gde-to 5 mkg/mL, no tut ia mogu oshibat`sia. Tak chto vopros k tem, kto vydelial RNA iz krovi: kakoi u vas byl vyhod?
Lady, 12.12.2002 09:50
Выделяла я РНК из крови. Просто модифицировала метод выделения из ткани, так как кита не было, а если проблем с покупкой китов нет то можно купить и использовать протокол выделения фирмы Promega.У них точно есть. Если же интерисует мой вариант, то сообщите мыло пришлю.
a26, 12.12.2002 16:35
Dear Lady, a kakoi u vas byl vyhod? Moi iznachal`nyi vopros: mojno li vydelit` 1-2 mkg RNA iz 0.1-0.3 mL krovi? [ 12-12-2002: Сообщение отредактировано: a26 ]
Kis, 12.12.2002 16:44
Нельзя. Если кровь нормальная - там столько просто нет, Промега не врет.
a26, 12.12.2002 17:28
То Кис: А сколько там есть? 1 мкг/мЛ, как "Промега" говорит, или побольше? [Текст переведён с транслита]
Kis, 13.12.2002 14:46
Ccылочка на Промеговскую рекламную статью. http://www.promega.com/pnotes/68/7381_23/7381_23.html Она там дают цифры от 0,75 до 5 мкг на мл. Реальная цифра - около 1,5 мкгмл (при условии нормальной крови, то есть 6000-8000 WBC на мкл человеческой крови). Или у вас не человеки ? А если человеки - то почему кубик не выкачать ?
Anonymous, 13.12.2002 15:12
А чего бы, действительно, не взять кубик, выделить лейкоциты на фиколле (или декстране), разболтать их в 200 мкл физраствора и выделять себе по методике Promega?
a26, 13.12.2002 17:44
Da net, u nas myshi, prichem ih malo, tak chto ubivat` ih nel`zia, poka potomstvo ne dadut. Tak chto mojem vziat` 100-300 mkL krovi, a eto, navernoe, 0.1-0.5 mkg RNA. Poprobuem poiti drugim putem: eta RNA nujna dlia RT (reverse transcription). Ia obychno beru na RT rxn 1 mkg RNA, no slyshala, chto est` metodiki, v kotoryh berut men`she RNA. Kto-nibud` eto delal? Mojet chto-nibud` podskazat`? Ia poka tol`ko nashla opisaniia kitov, v kotoryh skazano: vydelite RNA nashim metodom, rastvorite ee v 60 mkL i voz`mite 5 mkL na RT rxn? A v mkg eto skol`ko?
EKalina, 13.12.2002 20:33
Можно брать и меньше. Только надо потренироваться работать с РНК вслепую (осадок меньше 1мкг плохо или вовсе не видно). Только потом разведите свою реакцию не так сильно, как обычно. Или циклов придется делать побольше. А с каким ферментом работаете? Я бы порекомендовала SuperscriptII, так как он обычно дает больший выход кДНК, чем обычный MuLV или AMV.
a26, 13.12.2002 21:37
Vse, ia nashla: dlia Supercript poidet ot 1 pg RNA/rxn. Vsem spasibo, temu mojno zakryt`.
Veshka, 13.12.2002 23:01
Суперскриптом 2 как-то раз с ходу отревертировал _относительно_ abundant RNA из ~100 тысяч абсолютно дохлых, но лежавших в заморозке клеток. А в качестве кэрриера годится PelletPaint.
gregTS, 15.12.2002 01:18
А че такое этот PelletPaint и где его берут?
Veshka, 15.12.2002 02:44
Берут его в Новаджине, в Москве можно через химмед. Сходите в новаджин и посмотрите. Вещь весьма удобная.
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