做RNA的注意事项
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RNA WORK:
RNA work is not fun. Although as a molecule, it is just as robust and sturdy as DNA (a little more even, since it can form some pretty fancy tertiary structures), it suffers because the nucleases (RNases) that can destroy it ARE very stable molecules.
RNases are also EVERYWHERE.
They don’t require divalent cations as cofactors.
Unharmed by autoclaving procedures or exposure to temperatures as high as 150C!!!
Because of this, you have to take the following precautions.
MUST USE ASEPTIC TECHNIQUE. you quite seriously want to be as ANAL as possible. Segregate all your equipment, your space, even yourself if you have do. Different people exhibit different levels of care, but a general rule is to be as careful as possible. REMEMBER, RNases are EVERYWHERE!!
So use disposable sterile plastic wear (polypropylene), and if you have to use glassware treat it accordingly
i.e. Glassware-> cleaned with detergent/thoroughly rinsed and ovenbaked at >250C for >4hrs.
Show O/H DEPC treated Barbie doll
DEPC is a very useful reagent for making buffers and equipment RNase free. Essentially, you mix some DEPC with your buffer, let it sit, and get rid of the DEPC before using your liquid. For equipment, you immerse it in a similar solution and let the DEPC evaporate away.
WHAT DOES IT DO? DEPC results in a covalent modification of nucleases rendering them inactive. Histidine modifier – imine -> carbonate >NH >N-CO3
Things to consider when using DEPC: (comes as a liquid/ can buy at different percentages)
stinks. It is an organic solvent. Handy b/c you can tell its presence by its smell. Work in fumehood when treating water/buffers or glassware. Usually let your buffer+DEPC or equipment/glassware incubate for >12hrs. Then leave it in fumehood O/N with lid slightly ajar SO THAT FUMES ESCAPE. OR AUTOCLAVE the bugger (if possible).
DEPC in contact with water creates CO2 and Ethanol. If not careful and kept in sealed container can lead to excessive pressure buildup -> this is why you hear horror stories about it being explosive (not fireball explosive -> more like glass shattering explosive). Haven't personally heard of any incidents but just be careful when using.
Can't treat Tris solutions with DEPC . Tris is full of amines that DEPC will spend more of it's time modify the wrong thing
Some other notes when treating glassware or apparatus.
OR use immerse in fresh DEPC solution O/N (~12hrs). (you know its there if it stinks)
Then autoclave for 15min to remove residual DEPC.
Alternatively (gel box cleaning can be cleaned with 0.5% SDS. Rinsed with water, dried with ethanol. Filled with 3% hydrogen peroxide (10min). rinse thoroughly with Rnase free dH2O.
Chloroform also denatures Rnases, (rinse with chloroform). Can be quite harmful to plasticware.
GibcoBRL. Rnase AWAY. Reagent $100 per 250ml. Can wipe apparatus clean. Not harmful to plastics.
FINAL POINTERS:
ALWAYS WEAR GLOVES!! Good idea to change them periodically (maybe go through 2 to 3 sets today)
Once lysis has proceeded and RNA is exposed -> keep things COLD. RNases aren’t happy at 0-4C
ISOLATION OF RNA:
General procedure:
We are extracting RNA from plant material using a product called Trizol (available from Gibco BRL). Although this reagent is a proprietory product, it is most likely based on the familiar Phenol+Guanidium Thiocyanate procedure.
Guanidium thiocyanate is often classed as a denaturant although it is also considered a chaotrophic salt (sucks water)
This methods works in an analogous fashion to phenol chloroform extraction except that you also want to get your DNA to go into the phenol layer (therefore, you are left with just RNA in the the aqueous phase). This Trizol business works because RNA is still water soluble in a high molar guanidium thiocyanate solution whereas proteins and DNA is not. Consequently, the insoluble components will tend to go to the organic phase.
ALTERNATE PROCEDURE:
Another way of getting RNA is to utilize the fact that your RNA resides in the cytoplasm whilst the DNA resides in the nucleus (at least for eukaryotes). Consequently, an option is to first treat the cell with a "gentle" detergent to lyse the cell membrane but leave the nuclear membrane intact. Examples of common detergents used for this purpose are Triton X-100 and NP-40 (these two are almost identical). NOTE: The biochemistry and behaviour of detergents is very complicated. CONSEQUENTLY when dealing with a detergent, it is a good idea to follow the procedures given rather than playing around too much. Detergents have many attributes that affect their effectiveness. Dependant on their existence as free form particles or micelle complexes (kinda like a vesicle). Detergents forming micelles don't work as well and micelle formation is very sensitive to both temperature and concentration effects which vary enormously from detergent to detergent.
NOTES REGARDING NORTHERNS:
If you plan on transfering RNA over to a membrane, you have to go to extra efforts to get your RNA linear (RNA loves to form tertiary structures which will make sticking to the membrane difficult). SO… if you use RNA, it is a good idea to include a denaturant step (i.e. + formaldehyde, or use DMSO + glyoxal. (we have omitted this step, which may explain why its never works very well).
(For RNA work : We used cappilary action to transfer RNA that was treated with formaldehyde and run on a MOPS system gel - can't use Tris because of DEPC problem)
MORE STUFF ON RNA: (RNA techniques)
Interested in RNA processing.
Interested in mRNA amounts, what is in vivo translation rate
Interested in making a cDNA library.
Need to concern yourself with the quality of RNA prep. Total RNA is ~
%80 rRNA
%15 tRNA
<5% mRNA
most people concerned about mRNA (although some study rRNA as an evolutionary marker since the rRNA moelcules are very conserved from species to species -> use it to gage evolutionary trees).
Generally, the cleaner the mRNA prep the easier your life is gonna be. BUT getting purified mRNA sample is difficult in itself.
Almost all mRNA have poly A tails which is a very useful characteristic that many mRNA techniques take avantage of... Problem is that Rnase degradation is an even bigger concern since, these nucleases tend to chew the ends of RNA first. Examples of using the polyA tail.
YOU can use oligo dT affinity chromatography to purify your mRNA.
Can use the polyA region as a primer binding site for reverse transcriptase or PCR experiments.
GOLDEN RULE for mRNA work is as follows: Most techniques will still work with a total RNA prep, but you will get MUCH better results if you go to the effort of purifying your mRNA first.
(i.e. Making cDNA library strongly suggests you use a purified mRNA prep.)
See HO of techniques
S1 analysis
Rnase protection
RT PCR for mRNA quantitatioundefined~Kbr_~H~M~2~1~Kbr_~H~M~2~1Reverse_transcriptase~I_some_specifics~I~Kbr_~H~M~2~1NOTE_that_the_procedure_itself_is_very_straightforward._Difficulties_arise_primarily_because_of_the_RNA_prep.~Kbr_~H~M~2~1The_enzyme~I_Number_of_variants_available._AMV_~Aavian_myeloblastosis_virus~B~E_MMLV_~Amoloney_murine_leukemia_virus~B~E_HIV_~Ahuman_immunodeficiency_virus~B~E_and_other_various_proprietory_enzymes._~ASome_newer_ones_are_also_heat_stable_which_means_you_can_incubate_at_higher_temperatures_~F~8gt~J_therefore_more_stringent~E_more_specific_conditions~B~Kbr_~H~M~2~1~Kbr_~H~M~2~1unit_designation_is_usually_quite_complicated_~Arelates_the_amount_of_incorporated_labeled_nucleoide_in_the_reaction_under_specific_temperatures_and_specific_reaction_times_~F_basically_just_follow_instructions~B~Kbr_~H~M~2~1~Kbr_~H~M~2~1Three_basic_functions~I_~A1~B_RNA~Fdependent_DNA_polymerase~E_~A2~B_hybrid_dependant_ribonuclease_~ARNaseH~B_and_~A3~B_DNA_dependant_DNA_polymerase.~Kbr_~H~M~2~1~Kbr_~H~M~2~1Oligo_dT_primer_usually_a_minimum_of_12_nucleotides_in_length._Commonly_in_the_12~F20_nucleotide_range.~Kbr_~H~M~2~1In_prokaryotes_~F_common_idea_is_to_use_random_hexamer_primer_population.~Kbr_~H~M~2~1~Kbr_~H~M~2~1TWO_STEP_vs_ONE_STEP_RT~FPCR~I~Kbr_~H~M~2~1two_step_is_generally_more_reliable_as_you_have_the_option_of_tweaking_your_PCR_parameters_~Awill_talk_later~B._i.e._you_can_make_your_PCR_go_at_its_optimal_best~E_because_the_reaction_can_be_fine_tuned_independantly._Sometimes_you_have_to_do_two_step_because_the_RT_you_use_prefers_Mn2~D_as_a_cofactor._More_likelihood_for_cross_contamination_since_there_are_more_steps_~F_this_can_be_a_problem_if_your_mRNA_is_in_very_low_amounts_and_the_sensitivity_of_your_assay_is_high.~Kbr_~H~M~2~1~Kbr_~H~M~2~1one_step_is_quicker~E_less_work_BUT_also_less_reliable_because_your_PCR_reaction_conditions_are_restricted_to_the_same_conditions_used_in_your_reverse_transcriptase_assay._i.e._cofactor_amounts_stay_the_same._Affect_of_your_PCR_primers_in_RT_assay~E_etc_etc_etc._One_step_works_because_the_two_enzymes_~ART_and_the_heat_stable_DNA_pol_work_at_different_temperatures~B.
RNA work is not fun. Although as a molecule, it is just as robust and sturdy as DNA (a little more even, since it can form some pretty fancy tertiary structures), it suffers because the nucleases (RNases) that can destroy it ARE very stable molecules.
RNases are also EVERYWHERE.
They don’t require divalent cations as cofactors.
Unharmed by autoclaving procedures or exposure to temperatures as high as 150C!!!
Because of this, you have to take the following precautions.
MUST USE ASEPTIC TECHNIQUE. you quite seriously want to be as ANAL as possible. Segregate all your equipment, your space, even yourself if you have do. Different people exhibit different levels of care, but a general rule is to be as careful as possible. REMEMBER, RNases are EVERYWHERE!!
So use disposable sterile plastic wear (polypropylene), and if you have to use glassware treat it accordingly
i.e. Glassware-> cleaned with detergent/thoroughly rinsed and ovenbaked at >250C for >4hrs.
Show O/H DEPC treated Barbie doll
DEPC is a very useful reagent for making buffers and equipment RNase free. Essentially, you mix some DEPC with your buffer, let it sit, and get rid of the DEPC before using your liquid. For equipment, you immerse it in a similar solution and let the DEPC evaporate away.
WHAT DOES IT DO? DEPC results in a covalent modification of nucleases rendering them inactive. Histidine modifier – imine -> carbonate >NH >N-CO3
Things to consider when using DEPC: (comes as a liquid/ can buy at different percentages)
stinks. It is an organic solvent. Handy b/c you can tell its presence by its smell. Work in fumehood when treating water/buffers or glassware. Usually let your buffer+DEPC or equipment/glassware incubate for >12hrs. Then leave it in fumehood O/N with lid slightly ajar SO THAT FUMES ESCAPE. OR AUTOCLAVE the bugger (if possible).
DEPC in contact with water creates CO2 and Ethanol. If not careful and kept in sealed container can lead to excessive pressure buildup -> this is why you hear horror stories about it being explosive (not fireball explosive -> more like glass shattering explosive). Haven't personally heard of any incidents but just be careful when using.
Can't treat Tris solutions with DEPC . Tris is full of amines that DEPC will spend more of it's time modify the wrong thing
Some other notes when treating glassware or apparatus.
OR use immerse in fresh DEPC solution O/N (~12hrs). (you know its there if it stinks)
Then autoclave for 15min to remove residual DEPC.
Alternatively (gel box cleaning can be cleaned with 0.5% SDS. Rinsed with water, dried with ethanol. Filled with 3% hydrogen peroxide (10min). rinse thoroughly with Rnase free dH2O.
Chloroform also denatures Rnases, (rinse with chloroform). Can be quite harmful to plasticware.
GibcoBRL. Rnase AWAY. Reagent $100 per 250ml. Can wipe apparatus clean. Not harmful to plastics.
FINAL POINTERS:
ALWAYS WEAR GLOVES!! Good idea to change them periodically (maybe go through 2 to 3 sets today)
Once lysis has proceeded and RNA is exposed -> keep things COLD. RNases aren’t happy at 0-4C
ISOLATION OF RNA:
General procedure:
We are extracting RNA from plant material using a product called Trizol (available from Gibco BRL). Although this reagent is a proprietory product, it is most likely based on the familiar Phenol+Guanidium Thiocyanate procedure.
Guanidium thiocyanate is often classed as a denaturant although it is also considered a chaotrophic salt (sucks water)
This methods works in an analogous fashion to phenol chloroform extraction except that you also want to get your DNA to go into the phenol layer (therefore, you are left with just RNA in the the aqueous phase). This Trizol business works because RNA is still water soluble in a high molar guanidium thiocyanate solution whereas proteins and DNA is not. Consequently, the insoluble components will tend to go to the organic phase.
ALTERNATE PROCEDURE:
Another way of getting RNA is to utilize the fact that your RNA resides in the cytoplasm whilst the DNA resides in the nucleus (at least for eukaryotes). Consequently, an option is to first treat the cell with a "gentle" detergent to lyse the cell membrane but leave the nuclear membrane intact. Examples of common detergents used for this purpose are Triton X-100 and NP-40 (these two are almost identical). NOTE: The biochemistry and behaviour of detergents is very complicated. CONSEQUENTLY when dealing with a detergent, it is a good idea to follow the procedures given rather than playing around too much. Detergents have many attributes that affect their effectiveness. Dependant on their existence as free form particles or micelle complexes (kinda like a vesicle). Detergents forming micelles don't work as well and micelle formation is very sensitive to both temperature and concentration effects which vary enormously from detergent to detergent.
NOTES REGARDING NORTHERNS:
If you plan on transfering RNA over to a membrane, you have to go to extra efforts to get your RNA linear (RNA loves to form tertiary structures which will make sticking to the membrane difficult). SO… if you use RNA, it is a good idea to include a denaturant step (i.e. + formaldehyde, or use DMSO + glyoxal. (we have omitted this step, which may explain why its never works very well).
(For RNA work : We used cappilary action to transfer RNA that was treated with formaldehyde and run on a MOPS system gel - can't use Tris because of DEPC problem)
MORE STUFF ON RNA: (RNA techniques)
Interested in RNA processing.
Interested in mRNA amounts, what is in vivo translation rate
Interested in making a cDNA library.
Need to concern yourself with the quality of RNA prep. Total RNA is ~
%80 rRNA
%15 tRNA
<5% mRNA
most people concerned about mRNA (although some study rRNA as an evolutionary marker since the rRNA moelcules are very conserved from species to species -> use it to gage evolutionary trees).
Generally, the cleaner the mRNA prep the easier your life is gonna be. BUT getting purified mRNA sample is difficult in itself.
Almost all mRNA have poly A tails which is a very useful characteristic that many mRNA techniques take avantage of... Problem is that Rnase degradation is an even bigger concern since, these nucleases tend to chew the ends of RNA first. Examples of using the polyA tail.
YOU can use oligo dT affinity chromatography to purify your mRNA.
Can use the polyA region as a primer binding site for reverse transcriptase or PCR experiments.
GOLDEN RULE for mRNA work is as follows: Most techniques will still work with a total RNA prep, but you will get MUCH better results if you go to the effort of purifying your mRNA first.
(i.e. Making cDNA library strongly suggests you use a purified mRNA prep.)
See HO of techniques
S1 analysis
Rnase protection
RT PCR for mRNA quantitatioundefined~Kbr_~H~M~2~1~Kbr_~H~M~2~1Reverse_transcriptase~I_some_specifics~I~Kbr_~H~M~2~1NOTE_that_the_procedure_itself_is_very_straightforward._Difficulties_arise_primarily_because_of_the_RNA_prep.~Kbr_~H~M~2~1The_enzyme~I_Number_of_variants_available._AMV_~Aavian_myeloblastosis_virus~B~E_MMLV_~Amoloney_murine_leukemia_virus~B~E_HIV_~Ahuman_immunodeficiency_virus~B~E_and_other_various_proprietory_enzymes._~ASome_newer_ones_are_also_heat_stable_which_means_you_can_incubate_at_higher_temperatures_~F~8gt~J_therefore_more_stringent~E_more_specific_conditions~B~Kbr_~H~M~2~1~Kbr_~H~M~2~1unit_designation_is_usually_quite_complicated_~Arelates_the_amount_of_incorporated_labeled_nucleoide_in_the_reaction_under_specific_temperatures_and_specific_reaction_times_~F_basically_just_follow_instructions~B~Kbr_~H~M~2~1~Kbr_~H~M~2~1Three_basic_functions~I_~A1~B_RNA~Fdependent_DNA_polymerase~E_~A2~B_hybrid_dependant_ribonuclease_~ARNaseH~B_and_~A3~B_DNA_dependant_DNA_polymerase.~Kbr_~H~M~2~1~Kbr_~H~M~2~1Oligo_dT_primer_usually_a_minimum_of_12_nucleotides_in_length._Commonly_in_the_12~F20_nucleotide_range.~Kbr_~H~M~2~1In_prokaryotes_~F_common_idea_is_to_use_random_hexamer_primer_population.~Kbr_~H~M~2~1~Kbr_~H~M~2~1TWO_STEP_vs_ONE_STEP_RT~FPCR~I~Kbr_~H~M~2~1two_step_is_generally_more_reliable_as_you_have_the_option_of_tweaking_your_PCR_parameters_~Awill_talk_later~B._i.e._you_can_make_your_PCR_go_at_its_optimal_best~E_because_the_reaction_can_be_fine_tuned_independantly._Sometimes_you_have_to_do_two_step_because_the_RT_you_use_prefers_Mn2~D_as_a_cofactor._More_likelihood_for_cross_contamination_since_there_are_more_steps_~F_this_can_be_a_problem_if_your_mRNA_is_in_very_low_amounts_and_the_sensitivity_of_your_assay_is_high.~Kbr_~H~M~2~1~Kbr_~H~M~2~1one_step_is_quicker~E_less_work_BUT_also_less_reliable_because_your_PCR_reaction_conditions_are_restricted_to_the_same_conditions_used_in_your_reverse_transcriptase_assay._i.e._cofactor_amounts_stay_the_same._Affect_of_your_PCR_primers_in_RT_assay~E_etc_etc_etc._One_step_works_because_the_two_enzymes_~ART_and_the_heat_stable_DNA_pol_work_at_different_temperatures~B.
