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Sample to Insight

January 2018

RNeasy

Fibrous Tissue

Mini Handbook

For purification of total RNA
from heart, skeletal muscle,
aorta and other fiber-rich
tissues

Содержание RNeasy Fibrous Tissue

Страница 1: ...Sample to Insight__ January 2018 RNeasy Fibrous Tissue Mini Handbook For purification of total RNA from heart skeletal muscle aorta and other fiber rich tissues...

Страница 2: ...s 10 Determining the amount of starting material 10 Handling and storing starting material 12 Disrupting and homogenizing starting material 13 Protocol Purification of Total RNA Using the RNeasy Fibro...

Страница 3: ...010 HB 0485 002 Changes to comply with GHS regulation throughout the document December 2014 HB 0485 003 Removal of RNeasy Fibrous Midi Kit cat no 75742 including associated protocols general kit infor...

Страница 4: ...Buffer RLT 45 ml Buffer RW1 45 ml Buffer RPE concentrate 11 ml RNase Free Water 4 x 10 ml RNase Free DNase Set RNase Free DNase I lyophilized Buffer RDD RNase Free Water 1500 units 2 x 2 ml 1 5 ml Qu...

Страница 5: ...ated storage buffer The proteinase K is stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of the proteinase K we recommend storage at 2 8 C Intended Use Th...

Страница 6: ...ple preparation waste Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate Guanidine salts can form highly reactive compounds when combined with bl...

Страница 7: ...supernatant is removed The supernatant is mixed with ethanol and then centrifuged through an RNeasy Mini spin column where RNA binds to the silica membrane Traces of DNA that may co purify with the R...

Страница 8: ...RNeasy Fibrous Tissue Mini Handbook 01 2018 8 Figure 1 RNeasy Fibrous Tissue Mini workflow...

Страница 9: ...Disposable gloves Water bath or heating block capable of reaching 55 C Equipment for tissue disruption and homogenization see page 12 We recommend either the TissueRuptor II with TissueRuptor Disposab...

Страница 10: ...hown in Table 1 next page should be used so that the RNA binding capacity of the RNeasy Mini spin column is not exceeded When processing samples containing low amounts of RNA the maximum amount of sta...

Страница 11: ...ting material is incomplete RNA yields will be lower than expected even if the binding capacity of the RNeasy Mini spin column is not exceeded Table 2 Typical yields of total RNA Mouse rat tissue 10 m...

Страница 12: ...red at 90 to 65 C or immediately immersed in RNAlater RNA Stabilization Reagent or AllProtect Tissue Reagent at room temperature or immediately frozen in liquid nitrogen and stored at 90 to 65 C Other...

Страница 13: ...membrane and therefore significantly reduced RNA yields Disruption and homogenization of tissue samples can be carried out rapidly and efficiently using either the TissueRuptor II for processing samp...

Страница 14: ...stainless steel beads of 5 mm mean diameter For guidelines on using the TissueLyser II refer to the TissueLyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads...

Страница 15: ...starting material we recommend starting with no more than 10 mg tissue Depending on RNA yield and purity it may be possible to use up to 30 mg tissue in subsequent preparations IMPORTANT Do not overlo...

Страница 16: ...e continuing with step 5 Avoid prolonged incubation which may compromise RNA integrity Important Do not vortex reconstituted DNase I DNase I is especially sensitive to physical denaturation Mixing sho...

Страница 17: ...tion in step 6 2 If using the TissueLyser II add one stainless steel bead 5 mm mean diameter per 2 ml microcentrifuge tube not supplied If working with tissues that are not stabilized in RNAlater RNA...

Страница 18: ...suitably sized vessel Add 300 l Buffer RLT Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes 6 Place the tip of the disposable probe into th...

Страница 19: ...10 min 15 Centrifuge at 15 25 C for 3 min at 10 000 x g A small pellet of tissue debris will form sometimes accompanied by a thin layer or film on top of the supernatant 16 Pipet the supernatant appro...

Страница 20: ...ut not the collection tube Proceed to step 24 21 Add 10 l DNase I stock solution to 70 l Buffer RDD Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of...

Страница 21: ...hrough Otherwise carryover of ethanol will occur 26 Optional Place the RNeasy Mini spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Close th...

Страница 22: ...aterial It is essential to use the correct amount of starting material see page 10 and protocol c Centrifugation temperature too low The centrifugation temperature should be 15 25 C Some centrifuges m...

Страница 23: ...sure that tissue starting material samples are properly stabilized and stored in RNAlater RNA Stabilization Reagent for details see the RNAlater Handbook For frozen tissue samples ensure that they wer...

Страница 24: ...he RNeasy Mini spin column membrane by centrifuging at 8000 x g 10 000 rpm for 2 min at 15 25 C After centrifugation carefully remove the column from the collection tube so that the column does not co...

Страница 25: ...uring pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working wit...

Страница 26: ...e use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight...

Страница 27: ...fers and decomposes rapidly into ethanol and CO2 When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine...

Страница 28: ...the QIAxpert QIAxcel system or Agilent 2100 bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure significance A260 readings should be great...

Страница 29: ...e calculation involved in RNA quantification is shown below Volume of RNA sample 100 l Dilution 10 l RNA sample 490 l 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml...

Страница 30: ...an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 28 To assess the purity of RNA A260 A280 we recommend using the QIAxpert The QIAxpert is an...

Страница 31: ...designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible see www qiagen com GeneGlobe For real time RT PCR assays where amplification of...

Страница 32: ...RNA samples and allows easy interpretation of sample integrity The RIS is a value from 1 to 10 where a value of 10 indicates completely intact RNA Similarly the Agilent 2100 bioanalyzer offers an RNA...

Страница 33: ...rting Prepare DNase I stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase I 1500 Kunitz units in 550 l of the RNase free water provided To avoid loss...

Страница 34: ...e 87 5 l RNA eluate 10 l Buffer RDD 2 5 l DNase I stock solution 3 Make the volume up to 100 l with RNase free water The reaction volumes can be doubled if necessary to 200 l final volume 4 Incubate o...

Страница 35: ...A Stabilization Reagent 50 ml For stabilization of RNA in 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent 76104 RNAlater RNA Stabilization Reagent 250 ml For stabilization of RNA i...

Страница 36: ...acked on the TissueLyser II 69984 TissueLyser Single Bead Dispenser 5 mm For dispensing individual beads 5 mm diameter 69965 Stainless Steel Beads 5 mm 200 Stainless Steel Beads suitable for use with...

Страница 37: ...nts are licensed for one time use and may not be reused refurbished or resold 4 QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 5 The purchaser...

Страница 38: ...RNeasy Fibrous Tissue Mini Handbook 01 2018 38 Ordering www qiagen com shop Technical Support support qiagen com Website www qiagen com 1112114...

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