the equivalent of 1 od600 of cells should produce at least 1g of total rna and a minimum of 30 g is recommended for the procedure .
resuspend cells in lysis buffer solution consisting of 0.3 m buffered na - acetate ( ph 4.5 ) and 10 mm edta .
vortex with an equal volume of na - acetate - saturated phenol / chloroform ( ph 4.5 ) three times each for 30 s. be sure to always keep samples on ice between vortexing . buffered acetate can be prepared from naoac and hoac while acetate saturated phenol / chloroform can be prepared by mixing 1 part 5 m buffered acetate ( ph 4.5 ) with 9 parts phenol / chloroform .
note : total rna isolation is carried out under mildly acidic conditions and on ice to avoid deacylation of charged trna .
after the second extraction precipitate the rna by adding 2.7x volumes of ethanol and centrifuging at 18,600 rfc for 30 minutes at 4c .   resuspend rna pellets in lysis buffer solution and then precipitate with ethanol a second time .   finally , resuspend the rna in a solution containing 10 mm buffered acetate ( ph 4.5 ) and 1 mm edta for subsequent treatment
heat e. coli trna standards at 10.5 m to 85c in 45 mm tris - cl ph 7.5 for 2 minutes .
add mgcl2 to a final concentration of 20 mm and incubate at 37c for 5 minutes .
add the synthetase reaction mix so the final reaction contains 5 m trna , 60 mm tris - hcl ( ph 7.5 ) , 12.5 mm mgcl2 , 10 mm kcl , 3 mm dithiothreitol , 1.5 mm atp , 1 mm spermine , 1 mm of the respective amino acid , and 4.2 units/l e. coli aminoacyl - trna synthetase mix .
add an equal volume of 0.5 m buffered acetate ( ph 4.5 ) and extract with acetate - saturated phenol / chcl3 at ph 4.5 .
precipitate the trna standards with 2.7x volumes of ethanol and centrifuging at 18,600 rfc for 30 minutes at 4c .  
resuspend the standards in 50 mm buffered acetate ( ph 4.5 ) with 1 mm edta and store at 80c for up to one month .
for the charged trna sample incubate total rna at a concentration of 0.1 g/l with 0.066 m each e. coli trna standards ( i.e. 0.67 pmole each standard per g total rna ) and 100 mm buffered acetate ( ph 4.5 ) in the presence of 50 mm naio4 for 30 minutes at room temperature . for the control total trna sample use 50 mm nacl in place of naio4 . remember to dilute the rna only with buffered solutions to preserve charging .   to quench the reaction add glucose to 100 mm and incubate at room temperature for 5 minutes . in order to remove any remaining naio4 from the sample
for best results equilibrate the column first by running 200 mm buffered acetate buffer ( ph 4.5 ) through it prior to applying your sample .
precipitate the sample by adding buffered acetate ( ph 4.5 ) to a final concentration of 133 mm and nacl to a final concentration of 66 mm and 2.7x volumes of ethanol .
this is necessary only if the pellet looks significantly different than the control pellet of the same sample .
for example a significantly bigger or more diffuse pellet . if a second ethanol precipitation is required resuspend pellet in 50 mm acetate buffer ph 4.5 and 200 mm nacl before the addition of ethanol .   for deacylation
the trna samples are resuspended in 50 mm tris - hcl ( ph 9 ) and incubated at 37 c for 30 min .
the reaction is neutralized by the addition of an equal volume of 50 mm buffered acetate ( ph 4.5 ) and 100 mm nacl .
both the control and oxidized samples should be run on agarose gels to check rna quality . to attach fluorescent oligo tags onto the trna 0.1 g/l
deacylated rna is incubated in 1x ligase buffer , 15% dmso , 4 m cy3- or cy5-containing oligonucleotides , 0.5 units/l t4 dna ligase , and yeast exo - phosphatase ( 5,000 units/l ) at 16c overnight ( over 16 h ) .
the exo - phosphatase is necessary only for samples obtained from yeast and can be omitted if this protocol is used for e. coli or human samples .  
after ligation samples are mixed with 4 volumes of 50 mm koac ( ph 7 ) , 200 mm kcl , and then extracted with an equal volume of phenol / chloroform . following extraction of the aqueous phase , rna preparations are precipitated with ethanol and resuspended in water to approximately 0.1 g/l .
ligation efficiency can be assessed by running 5 - 10% of the samples on 12% polyacrylamide gels containing 7 m urea and visualized with a fluorescent gel scanner .
this page analysis is also useful to determine the amount of oxidized and control samples needed for microarray hybridization .
a good ligation result should show ~10% or more of the cy3/cy5 containing oligonucleotide being ligated to the trna .
for samples with good labeling efficiency , 0.1 - 0.5 g total rna per sample is used for array hybridization . prior
to hybridization microarray slides are boiled in distilled water for 1 - 2 minutes to remove unbound oligonucleotides .
cy3/cy5 labeled samples are combined with 140 l of hybridization buffer and salmon sperm dna to a final concentration of 140 g / ml and poly(a ) to a final concentration of 70 g / ml . a hybridization program , ( table 1 ) , is run on an automatic array hybridizer . for trna work
, it is highly recommended that an automatic hybridization machine be used since manual hybridization produces high background . after the program is complete
manually wash the slides with wash 3 solution at least twice by gently shaking in a 50 ml conical tube for 3 - 5 minutes .
slides can be dried by centrifuging at a low speed , less than 200 rfc , for 5 - 10 minutes .
it is important to keep the slides out of the light since exposure to light will bleach the fluorophores .
after the slides are dried they should be scanned immediately for optimal results ; if necessary they can be saved for several days in a dry dark place at room temperature .
when isolated properly the total rna sample should produce a very clean spectrum with an od260:od280 ratio near 2 .
there should be no detectable protein or phenol contamination . when run on 1% agarose gels , a strong trna band should be observed with other possible nucleic acid bands varying between organisms .
if a sample is noticeably weaker than other samples or smearing is observed , samples should not be used for microarrays .
microarrays should have very low background which is an order of magnitude lower than the weakest trna probe .
this can usually be fixed by preparing fresh wash solutions and thoroughly washing all equipment and tubing to remove residual and precipitated sds . 

the equivalent of 1 od600 of cells should produce at least 1g of total rna and a minimum of 30 g is recommended for the procedure .
resuspend cells in lysis buffer solution consisting of 0.3 m buffered na - acetate ( ph 4.5 ) and 10 mm edta .
vortex with an equal volume of na - acetate - saturated phenol / chloroform ( ph 4.5 ) three times each for 30 s. be sure to always keep samples on ice between vortexing . buffered acetate can be prepared from naoac and hoac while acetate saturated phenol / chloroform can be prepared by mixing 1 part 5 m buffered acetate ( ph 4.5 ) with 9 parts phenol / chloroform .
note : total rna isolation is carried out under mildly acidic conditions and on ice to avoid deacylation of charged trna .
after the second extraction precipitate the rna by adding 2.7x volumes of ethanol and centrifuging at 18,600 rfc for 30 minutes at 4c .   resuspend rna pellets in lysis buffer solution and then precipitate with ethanol a second time .   finally , resuspend the rna in a solution containing 10 mm buffered acetate ( ph 4.5 ) and 1 mm edta for subsequent treatment
heat e. coli trna standards at 10.5 m to 85c in 45 mm tris - cl ph 7.5 for 2 minutes .
add mgcl2 to a final concentration of 20 mm and incubate at 37c for 5 minutes .
add the synthetase reaction mix so the final reaction contains 5 m trna , 60 mm tris - hcl ( ph 7.5 ) , 12.5 mm mgcl2 , 10 mm kcl , 3 mm dithiothreitol , 1.5 mm atp , 1 mm spermine , 1 mm of the respective amino acid , and 4.2 units/l e. coli aminoacyl - trna synthetase mix .
add an equal volume of 0.5 m buffered acetate ( ph 4.5 ) and extract with acetate - saturated phenol / chcl3 at ph 4.5 .
precipitate the trna standards with 2.7x volumes of ethanol and centrifuging at 18,600 rfc for 30 minutes at 4c .  
resuspend the standards in 50 mm buffered acetate ( ph 4.5 ) with 1 mm edta and store at 80c for up to one month .
for the charged trna sample incubate total rna at a concentration of 0.1 g/l with 0.066 m each e. coli trna standards ( i.e. 0.67 pmole each standard per g total rna ) and 100 mm buffered acetate ( ph 4.5 ) in the presence of 50 mm naio4 for 30 minutes at room temperature . for the control total trna sample use 50 mm nacl in place of naio4 . remember to dilute the rna only with buffered solutions to preserve charging .   to quench the reaction add glucose to 100 mm and incubate at room temperature for 5 minutes . in order to remove any remaining naio4 from the sample
for best results equilibrate the column first by running 200 mm buffered acetate buffer ( ph 4.5 ) through it prior to applying your sample .
precipitate the sample by adding buffered acetate ( ph 4.5 ) to a final concentration of 133 mm and nacl to a final concentration of 66 mm and 2.7x volumes of ethanol .
this is necessary only if the pellet looks significantly different than the control pellet of the same sample .
for example a significantly bigger or more diffuse pellet . if a second ethanol precipitation is required resuspend pellet in 50 mm acetate buffer ph 4.5 and 200 mm nacl before the addition of ethanol .   for deacylation
the trna samples are resuspended in 50 mm tris - hcl ( ph 9 ) and incubated at 37 c for 30 min .
the reaction is neutralized by the addition of an equal volume of 50 mm buffered acetate ( ph 4.5 ) and 100 mm nacl .
both the control and oxidized samples should be run on agarose gels to check rna quality . to attach fluorescent oligo tags onto the trna 0.1 g/l deacylated rna
is incubated in 1x ligase buffer , 15% dmso , 4 m cy3- or cy5-containing oligonucleotides , 0.5 units/l t4 dna ligase , and yeast exo - phosphatase ( 5,000 units/l ) at 16c overnight ( over 16 h ) .
the exo - phosphatase is necessary only for samples obtained from yeast and can be omitted if this protocol is used for e. coli or human samples .  
after ligation samples are mixed with 4 volumes of 50 mm koac ( ph 7 ) , 200 mm kcl , and then extracted with an equal volume of phenol / chloroform . following extraction of the aqueous phase , rna preparations are precipitated with ethanol and resuspended in water to approximately 0.1 g/l .
ligation efficiency can be assessed by running 5 - 10% of the samples on 12% polyacrylamide gels containing 7 m urea and visualized with a fluorescent gel scanner .
this page analysis is also useful to determine the amount of oxidized and control samples needed for microarray hybridization .
a good ligation result should show ~10% or more of the cy3/cy5 containing oligonucleotide being ligated to the trna . for samples with good labeling efficiency , 0.1 - 0.5 g total rna per sample
prior to hybridization microarray slides are boiled in distilled water for 1 - 2 minutes to remove unbound oligonucleotides .
cy3/cy5 labeled samples are combined with 140 l of hybridization buffer and salmon sperm dna to a final concentration of 140 g / ml and poly(a ) to a final concentration of 70 g / ml . a hybridization program , ( table 1 ) , is run on an automatic array hybridizer . for trna work , it is highly recommended that an automatic hybridization machine be used since manual hybridization produces high background . after the program is complete manually wash the slides with wash 3 solution at least twice by gently shaking in a 50 ml conical tube for 3 - 5 minutes .
slides can be dried by centrifuging at a low speed , less than 200 rfc , for 5 - 10 minutes .
it is important to keep the slides out of the light since exposure to light will bleach the fluorophores .
after the slides are dried they should be scanned immediately for optimal results ; if necessary they can be saved for several days in a dry dark place at room temperature .
when isolated properly the total rna sample should produce a very clean spectrum with an od260:od280 ratio near 2 . there should be no detectable protein or phenol contamination . when run on 1% agarose gels , a strong trna band should be observed with other possible nucleic acid bands varying between organisms .
if a sample is noticeably weaker than other samples or smearing is observed , samples should not be used for microarrays .
microarrays should have very low background which is an order of magnitude lower than the weakest trna probe .
this can usually be fixed by preparing fresh wash solutions and thoroughly washing all equipment and tubing to remove residual and precipitated sds . 

we present a method for simultaneously determining the relative charging levels of all trnas at the genomic scale .
the protocol presented here has been optimized for s. cerevisiae , and it has also been successfully used to measure trna charging profiles in e. coli and human cell cultures .
it can be modified to accommodate any organism with known genome sequences ( allowing for annotation of all trna genes ) as long as total charged trna can be obtained .
 trna aminoacylation , or charging , levels can rapidly change within a cell in response to the environment[1 ] . 
 changes in trna charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response . 
 familiar examples are the stringent response in e. coli and the gcn2 stress response pathway in yeast ( [ 2 - 6 ] ) . 
 recent work in e. coli and s. cerevisiae have shown that trna charging patterns are highly dynamic and depends on the type of stress experienced by cells [ 1 , 6 , 7 ] . 
 the highly dynamic , variable nature of trna charging makes it essential to determine changes in trna charging levels at the genomic scale , in order to fully elucidate cellular response to environmental variations . in this review 
 we present a method for simultaneously measuring the relative charging levels of all trnas in s. cerevisiae . while the protocol presented here 
 is for yeast , this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including e. coli and human cell cultures[7 , 8 ] . 
Protocol
Part 1: Isolation of total charged tRNA
Part 2: Preparation of tRNA Standards
Part 3: Cy3/Cy5 labeling of tRNA
Part 4: Hybridization and Analysis of the Microarray
Part 5: Representative Results
Discussion
Disclosures