Здравствуйте, хотел поинтересоваться пробовал ли кто-нибудь анализировать микроРНК методом обратной транскрипции,?
Основная трудность у меня заключается в создании праймеров для исследования miR398 a,b,c.
Подскажите каким софтом лучше пользоваться, если кто-нибудь работал и делал что-то подобное прошу поделитесь опытом и прописями, для меня это новая область мало что известно...
заранее безгранично благодарен, не бейте если что не так

На самом деле ничего страшного тут нет. Я из любопытства разок попробовал - получилось. Софт не поможет, праймеры для обратной транскрипции, адаптор (для больше длины ампликона) и праймеры для ПЦР необходимо подбирать вручную. Следите за тем, чтобы РНК была полностью очищена, а также проверьте miR на наличие гомологий среди РНК. И вперед.

Скажите, многоуважаемый Derro, а Вы именно microRNA определяли?
Каким методом выделения RNA Вы пользовались?
Как можно без софта понять гомологична моя microRNA или нет?

определить гомологию без софта сложно. Я говорил про подбор праймеров. Я выделял РНК четырьмя методами: киты от киагена, тризол и Хомчинский. Плюс-минус идет везде.

Спсибо за подсказки.
Ещё есть небольшой вопрос.
Например у меня есть известная последовательность mRNA и в ней есть известная последовательность моей microRNA, но так как я буду делать обратную транскрипцию значит мне необходимо последовательность mRNA превратить в cDNA и уже к этой последовательности искать праймеры, правильно я понял?

http://www.pubmedcentral.nih.gov/picrender...95&blobtype=pdf

Привет, ОТ-ПЦР, насколько я понимаю, грубоват, и не позволит показать небольшую разницу в экспрессии, тем более если копийность итерисующей мир в ткани низкая, вы посмотрете может она там вообще не экспрессируеться ;-), нет плохо работает, на скорее всего эта мир оносится к семейству где разница между мир в 1 нуклеодит, так что ваши праймеры могут "ловить" всех членов семейства. Результат использовани таких праймеров может вызвать сомнения, если только не показать их 99% специфичность на определенную мир, и если адекватно сработают (-) и (+) контроли. ))))
Денис.

Да ОТ-ПЦР грубый метод, но к сожалению сейчас приходится работать именно таким методом. Денис, скажите как можно показать 99% специфичность на мою miR и как лучше сделать + - контроли ?, пока мне сложно понять как это будет работать. По мере продвижения эксперимента буду обязательно публиковать свои результаты на этой страничке.

обратную транскрипцию на микру-РНК длинной 21нт

Можно заказать такманскую пробу.

to Namelles
если люди подходят с такого "конца"
300-350 баков на контроль и их мир сеты тратить ....

Solian

Денис, не могу Вам отправиль личку что-то с форумом.
пишите на почту
biomaquinademuerte@gmail.com с уважение Павел!

тут метода с доращиванием микр полиаденелированием
http://www.plosone.org/article/info:doi/10...al.pone.0003148

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