Материал из Zbio

Перейти к: навигация, поиск

 Problem list:

  •  ?? samples in parallel
  • estimation of RNA concentration after purification
  • typical output
  • typical time

Mol.biology // Next-generation sequencing // RNA



NB! RNA: 1-10µg total in no more then 10µl for each reaction
NB! it is unpractical to process more than ?? samples in parallel

prepare in advance:

» 1L glass beaker
» magnetic mixer
» 37°C water bath
» magnetic rack
» RNase-free water (not DEPC-treated!)

  • sign 1.5ml LowBind tubes:
» _name_: three per each sample
» _1_name_: for first beads extraction
» _2_name_: for second beads extraction

[править] Hybridization

  1. prepare Hybridization mixture in 1.5ml LowBind tube (_name_/first):
    total RNA, 1-10µg<10µl
    RiboMinus Probe, 15pmol/µl10µl
    Hybridization Buffer100µl
  2. preheat to 75°C 500ml of water in 1L glass beaker with a stirring rod;
  3. incubate Hybridization mixture at 70-75°C for 5 min.[1];
  4. put glass beaker on magnetic mixer and allow the water to cool to 37°C for ~30 min.[2];
  5. while Hybridization mixture is cooling down, prepare beads;
  6. when temperature drops down to ~37°C, put tube with Hybridization mixture on 37°C water bath for at least 30min.

[править] Preparing Beads

  • should be done in advance upon arrival of RiboMinus kit:
» resuspend RiboMinus Magnetic Beads in its bottle by thorough vortexing
» aliquot 750µl of beads into 1.5ml LoBind tubes
» store at 4°C

  1. put 750µl aliquote of RiboMinus Magnetic Beads into magnetic rack for 1 min. Discard the supernatant;
  2. ○○   repeat two times H2O washing:
    • add 750µl RNase-free water to the beads and resuspend beads by slow vortexing
    • magnetic rack for 1 minute. Discard the supernatant
  3. resuspend beads in 750µl of Hybridization Buffer;
  4. transfer 250µl beads to a new tube (_2_name_) and keep this tube in 37°C water bath;
  5. place the tube with 500µl beads on magnetic rack for 1 min. Discard the supernatant;
  6. resuspend beads in 200µl of Hybridization Buffer and keep the tube in 37°C water bath (_1_name_)

[править] Removing rRNA

  • preheat magnetic rack in 37°C water bath, blot with paper towel before use

  1. pulse-spin tube with Hybridization mixture;
  2. transfer the sample (~120µl) to preheated _1_name_ tube. Mix well by pipetting;
  3. incubate at 37°C for 15 min. Gently mix the contents each 5 min.:
    » ________________
    » ________________
  4. magnetic rack for 1 min. Transfer supernatant into a new _name_/second tube[3];
  5. place tube with one-time extracted RNA in 37°C water bath;
  6. place _2_name_ tube with 250 µL beads on a magnetic rack for 1 min., aspirate and discard the supernatant;
  7. add to this tube one-time extracted RNA (~320µl). Mix well by pipetting;
  8. incubate the tube at 37°C for 15 minutes. Gently mix the contents each 5 min.:
    » ________________
    » ________________
  9. spin down briefly;
  10. magnetic rack for 1 min. Transfer supernatant (~320µl) into a new _name_/third tube[4];

[править] Ethanol Precipitation

  1. add (and mix after each component) to two-times extracted RNA:
    » 1µl of glycogen, 20µg/µl
    » 0.1x V (32µl) of AcONa, 3M, pH5
    » 2.5x V (880µl) of 100% Ethanol
  2. mix well and incubate at -20°C[5] for >30 min.;
  3. centrifuge max speed, 15 min. at 4°C;
  4. wash pellet twice with 250µl of 70% cold ethanol;
  5. air-dry the pellet for ~5 min.;
  6. resuspend RNA in 10µl RNase-free water;
  7. estimate RNA concentration:
    » start from <??µg of totalRNA: ???
    » start from ??µg<RNA<10µg of totalRNA: ???
  8. storage:
    » ice-bath for couple of hours
    » -20°C for 2-3 weeks
    » -80°C for long-term storage

[править] Reagents

» RiboMinus Eukaryote Kit for RNA-Seq: Invitrogen, #A10837-08
» Nuclease-free water, not DEPC treated: Ambion

[править] Notes

  • total RNA is hybridized with 5'-biotin labeled oligonucleotide probes for 18S, 28S, 5.8S, and 5S ribosomal RNA (2 probe types for each rRNA). The probes hybridize with highly conserved regions of rRNA (human, mouse, rat, drosophila, yeast — with zero mismatches). The rRNA/probe complex is removed with streptavidin-coated magnetic beads. The RiboMinus RNA sample is concentrated by ethanol precipitation.
  • typical output is 1µg from 10µg of total RNA (human tumor).
  • run time is
» one sample:
» ??? samples:
  • critical importance is to avoid beads drying.

  1. incubation at 70-75°C is fo denaturation of RNA
  2. slow cooling is important to promote specific hybridization
  3. the supernatant contains one-time extracted RiboMinus RNA
  4. the supernatant contains two-times extracted RiboMinus RNA
  5. Stratacooler box
Источник — «http://zbio.net/wiki/RiboMinus»

Личные инструменты


molbiol.ru  ·  redactor@molbiol.ru  ·  реклама

 ·  Викимарт - все интернет-магазины в одном месте  ·  Доска объявлений Board.com.ua  · 
--- сервер арендован в компании Hetzner Online, Германия ---
--- администрирование сервера: Intervipnet ---

molbiol.ru - методы, информация и программы для молекулярных биологов     Rambler's Top100 Rambler