mRNA Purification: Globin & rRNA Depletion Protocol for cDNA

[00:00:08][Music] with this protocol total RNA is hybridized with  DNA probes singlestranded DNA probes that target   your unwanted but abundant RNAs so we're talking  globin messenger RNA and ribosomal RNA this step   is required because globin messenger RNA is highly  abundant in total RNA extracted from blood samples   or if you cultured your samples with any amounts  of blood having these messenger RNAs in the system   makes it difficult to study messenger RNAs (mRNAs)  that are lowly expressed. as you may recall,   red blood cells contain hemoglobin. the globin  part is actually four (4) globin chains that are   joined together to form your globin; which is then  paired with your heme. Incidentally, the gene on   the... on your chromosomes, is chromosome 11 and  16 that provide this instruction to create globin.   the chains of globin are named by Greek letters.  there are seven Greek letters that are used;   there is alpha, beta, delta, gamma, epsilon,  zeta, and theta. so one strategy for enriching   for messenger RNAs mRNA) is to deplete all those  RNAs that are not messenger RNA. so in this case   you will deplete all your ribosomal RNAs. in  the cytosol of cells, you will find transcripts-   ribosomal RNA transcripts. so you will find 5S,  5.8S, 8s, 18s, 28s, ITS and ETSs and you can   target those and remove them. likewise you'll have  mitochondrial ribosomal RNA 12S and 16S. so you   can also remove them and so what you're left with  are messenger RNA transcripts only. however like   I said if you have blood in the system then you're  going to also have globin messenger RNAs and this   is where you will have probes that target those  globin messenger RNAs and eliminate them so that   you really only have messenger RNAs related to  the system that you're studying. 5s ribosomal RNAs   are located in the large ribosomal subunit. 5.8s  ribosomal RNAs are approximately 160 nucleotides   long and they are also part of the large ribosomal  subunit the 18S ribosomal RNA on the other hand   is part of the small ribosomal subunit it  consists of 1,870 nucleotides. there's also   the 28S ribosomal subunit which has approximately  5,000 nucleotides and it's also part of the large   ribosomal subunit. ITS stands for internal  transcribed spacer so these have approximately   200 to up to 3,000 nucleotides and they are found  as a precursor of the 45s preRNA transcript but   they are removed before ribosome assembly. then  there are ribosomal subunits the ETSs. these are   external transcribed spaces. these range from  200 to 2,000 nucleotides and they're found at   the five prime (5') and three prime (3') ends of  the precursor 45S preRNA transcript. Again, they   are removed before ribosome assembly but their  transcripts will be in the cytoplasm. a popular   alternative for enriching for messenger RNA is to  use short pieces (oligonucleotides) that enrich   for the messenger RNA only. so this oligo-based  messenger RNA enrichment this method selects for   only polyadenylated messenger RNAs and naturally  depletes ribosomal RNA and globin messenger RNA.   but just note that with this method you may have  some polyA remaining from globin messenger RNA.   Here's the protocol for enriching for messenger  RNAs and getting rid of globin and ribosomal   RNAs. so for the next... neb... neb next globin  and ribosomal RNA depletion kit, you're going to   get the neb next globin and RNA depletion solution  you're going to get the probe hybridization buffer   they'll also supply the thermostable RNase H.  you're going to get RNase H reaction buffer   and then a DNA I and DNA I reaction buffer. as  well as nuclease-free water. these items have to   be stored at minus 20 (-20oC) then the following  item has to be stored in the fridge you don't want   to freeze it. so you're going to be given the RNA  sample purification beads. there are other things   that the kit doesn't come with but you need to get  them. so you need to make 80% ethanol, you need to   get a micro centrifuge, a vortex, thermocycler, as  well as a magnetic rack and you need access to a   bioanalyzer or tape station for fragment analysis.  we'll need 200uL PCR tubes. so this protocol   you're using RNase H to deplete RNA that's going  to deplete globin messenger RNA. so apparently the   messenger RNA is... for globin you get HBA 1/2,  you get HB... HBD, HBM, HBG1 /2, HB... HBQ1 and   HBZ. These are globin messenger RNAs. Then you're  going to deplete cytoplasmic ribosomal RNAs. so   your 5S, 5.8S, 18S, 28S, ITS and ETSs. you're also  going to deplete mitochondrial ribosomal RNA so   12S and 16S. once you perform this depletion  of ribosomal RNA your sample is now suitable   for random prime complimentary DNA synthesis. not  the oligo (dT)- based synthesis. you can also use   the RNA that you deplete for RNA sequencing  as well as other downstream RNA analysis.   the RNA that you intend to use for depletion  has to be assessed by running the RNA sample   on an agent bioanalyzer. you can use the RNA 6000  nano or pico chip to determine the RNA integrity   number. Additionally, the RNA has to be free  of salt so no magnesium or guanidinium salts or   organic solvents; your phenol and ethanol should  be removed. the RNA has to be free of DNA. genomic   DNA is actually a common contaminant from RNA  preps and this is because it may be carried over   from the interphase if you use TRIzol or other  organic extractions or when the silica matrix   of your solid phase RNA purification methods, is  overloaded. this can be easily avoided by treating   the RNA with DNase I but then you have to remove  the enzyme, because any residual activity of DNase   I will degrade the single stranded DNA probes  necessary for your ribosomal RNA depletion.   after you treat with DNase I, you have to remove  the enzyme by phenol chloroform extraction and   ethanol precipitation. or you can use a silica  column such as with... the with... the common   column based assays... um purification. Before  purification, the RNA must be in nuclease-free   water. for the RNA input for depletion you can  use as little as 10 nano grams (ng) and as much   as 1 microgram (ug) and it has to be in a volume  of 10 micro Litres (ul) of nuclease-free water   and you have to quantify it by an RNA specific  dye-assisted fluorometric method. So the qubit   or ribogreen or a bioanalyzer. so the samples have  to be kept on ice throughout the process. so the   first step we will have our total RNA in nuclease  free water. Again we can use 10 ng to 1 ug. you   will need the globin and ribosomal RNA depletion  solution and the probe hybridization mix. so here   you can make a master mix by adding the globin  and ribosomal RNA depletion solution with the   probe hybridization buffer for as many samples as  you have and then add in 10 uL of your total RNA.   so once you add the hybridization buffer with the  globin and ribosomal RNA depletion solution, you   want to mix really well. so here it's recommended  you mix 10 times at least, because we've been   mixing... pipetting up and down for quite  some time, we need to spin it. to just briefly   centrifuge it to get all the liquid to settle back  into the tube. so now we need a thermal cycler.   we're going to heat up the sample to 95° C for 2  minutes then we're going to ramp down to 22° and   what you're going to do is set it to ramp down at  0.1° C every second. we're going to hold it at 22°   C for 5 minutes. and then you can just leave  it at 4° until you're ready. the program will   take around 15 to 20 minutes to complete and then  you want to briefly spin it down in a centrifuge,   in case there was some condensation and then place  it immediately on ice and we're going straight   into RNase H digestion. for this step we want to  grab our hybridized RNA - we need 50 uL of it. you   need your themostable RNase H. Then you need your  RNase H reaction buffer and some nuclease-free   water. just to make up to the volume... total  volume of 20 uL. again we want to mix thoroughly   so we're going to pipette it up and down at least  10 times. then we're going to briefly spin it in a   micro centrifuge just to collect everything nicely  to the... in the tube. we incubate the tube in a   preheated thermal cycler, this time we're going  to incubate for 30 minutes at 50° C with the lid   set to 55° after which we will hold at 4°C. after  this we want to briefly spin down again just to   collect everything nicely back into the tube and  place it on ice and proceed immediately to DNase   I digestion. in the next protocol we want to  digest DNA so here we're going to use our RNase   H treated RNA along with DNase I reaction buffer  and the DNase I enzyme. and what we're going to   do is make it up in a reaction volume of 50 uL.  so we... our input for the RNase H treated RNA is   20 uL. then we add the DNase I reaction buffer  at 5 uL. then we add the enzyme the DNase I at   2.5 uL and top up to 50 uL using nuclease free  water. again we want to mix really well so we we   will pipette up and down at least 10 times. then  because we've been moving the solution around,   we're going to briefly spin it just to collect  everything into the tube nicely before we incubate   it in a thermocycler for 30 minutes this time at  37° and we can have the lid off or if we're having   the lid on then put it on 40° C. After this 37°C  incubation we want to just briefly spin down again   just to collect everything into the tube in case  there was condensation and we're going to proceed   immediately to RNA purification. for this step we  either use an Agencourt RNA cleanup XP beads or   the RNA sample purification beads. so what we want  to do is just vortex either of these reagents so   that they're resuspended. then we're going to add  90 uL which turns out to be 1.8 times the volume   so 90 uL of beads to the RNA samples and we mix  this again very thoroughly; so at least 10 times   we need to incubate the tubes for 15 minutes  on ice. and this allows the RNA to bind to the   beads after this 15-minute incubation we pop it  on a magnetic rack to separate the beads from the   supernatant. once the solution runs clear - this  may take about 5 minutes -you want to carefully   remove and discard the supernatant. you want  to be careful not to disturb the beads because   these beads now contain the RNA. so you go ahead  and add 200 uL of freshly prepared 80% ethanol   to the tube, while it's on the magnetic rack and  again we're going to leave this to incubate for 30   seconds and then carefully remove and discard the  supernatant. we'll repeat the wash one additional   time then we go ahead and completely remove  residual ethanol, air dry the beads for about   5 minutes before proceeding to elute the RNA  from the bead with 7 ul of nuclease free water.   once we add the seven uL of nuclease free water  we want to once again mix really well. so we're   going to pipette up and down at least 10 times  and briefly spin the tube. once we've mixed   the nuclease free water with the beads we want to  leave it for about 2 minutes at room temperature.   then we're going to place the tubes back  on the magnetic rack until the solution is   clear and once it's clear we remove 5 uL  of the supernatant and this contains the   RNA this time. and we're going to transfer  it to a nuclease free tube. the RNA is now   ready for use in complementary DNA (cDNA)  library synthesis or for RNA seq or other   downstream applications. or you can store  it at minus 80oC for long-term storage.

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