AquaPreserve User Manual - MultiTarget Pharmaceuticals
Contents
1. the stabilized blood samples should be stored at 80 C Do not store AquaPreserve ProSink stabilized blood samples at 20 C because it reduces RNA yield significantly by unknown mechanism Fresh blood samples should be immediately stabilized with AquaPreserve and ProSink upon arrival at the laboratory Un stabilized frozen blood samples received should be stored at 80 C or at 20 C 4 Extract the DNA and RNA 1 To extract DNA RNA from AquaPreserve stabilized blood At the time of DNA RNA extraction simply thaw the AquaPreserve and ProSink stabilized blood sample centrifuge to pellet the proteins save the pellet for protein recovery if desired and recover the clear lysate and then precipitate the DNA RNA with 1 volume of isopropanol 2 To extract DNA RNA from frozen blood If the blood sample is contained in a tube 3x of the sample volume simply add one volume of AquaPreserve to the frozen blood shake and vortex vigorously to thaw the blood in AquaPreserve If the frozen blood is contained in the original vacutainer the bottom of the vacutainer may be cut off so that the frozen blood pellet may be pushed out into a 50 ml conical tube preloaded with one volume of AquaPreserve Shake and vortex vigorously to thaw the frozen blood in AquaPreserve Centrifuge the crude lysate at 14 000 xg for 5 min to pellet the debris and add 0 5 volume of ProSink with respective to the volume of AquaPreserve to the lysate shake and vortex2. shake holding the tube in your hand while shaking it will help the AquaPreserve solution penetrating into the frozen blood to thaw the blood Do not thaw the frozen blood without mixing with AquaPreserve or the RNA will be degraded during blood thawing However for blood DNA extraction only the blood sample should be thawed incubated at 22 C for 20 min to degrade the RNA prior to mixing with AquaPreserve Incubate at 22 C for 15 min Touch vortex a few times and centrifuge at 14 000 xg for 5 min to pellet the debris 2 Pellet the proteins Add 0 125 ml of ProSink to the lysate Invert the tube and touch vortex a few times to mix the lysate and ProSink without dislodging the debris pellet Incubate at 22 C for gt 30 min Blood DNA is now stable at 4 22 C for months and blood RNA is stable at 4 C for 2 weeks and 22 C for 7 days Centrifuge at 14 000 xg for 5 min to pellet the proteins 3 Pellet the DNA RNA Transfer the supernatant 0 7 ml to a new 1 5 ml microfuge tube If needed centrifuge the clear lysate again to remove any carried over debris Add 1 vol 0 7 ml of isopropanol Touch vortex a few times to mix well Centrifuge at 14 000 xg for 5 min to pellet the DNA RNA 4 Rinse the DNA RNA pellet Decant to discard the supernatant or save it for small molecules analysis Gently shoot 50 isopropanol from a squirt bottle to fill up the tube Do not to shoot the alcohol solution directly onto the pellet and decant t
3. with one of them complementary to the unique tailed region of the RT primer to amplify the cDNA Hurteau and Spivack mRNA specific reverse transcription polymerase chain reaction from human tissue extracts Anal Biochem 2002 Aug 15 307 2 304 15 and Chen et al Real time quantification of microRNAs by stem loop RT PCR Nucleic Acids Research 2005 33 20 e179 especially when intron spanning is unavailable In any case you should always include a no RT control in your amplification to confirm that your primers do not amplify the contaminating genomic DNA MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com
4. 0 xg for 5 min to pellet the proteins Decant to discard the supernatant Immediately add 100 ul of ProMelt 1150 order separately to the wet protein pellet Pipet and vortex to solubilize the proteins Centrifuge to pellet any insoluble The protein solution may be loaded directly to SDS PAGE Note If the buffy coat contains large amount of RBC you may need to use 2 volumes of AquaPreserve for the extraction or try various volume of ProSink 9030 for protein precipitation to reduce hemoglobin contamination of the DNA RNA pellet MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction Frequently Asked Questions Please read through these questions carefully The answers provide additional tips and useful information for the successful use of AquaPreserve 1 How should I store the AquaPreserve solution It may be stored at 22 C for 12 months If AquaPreserve becomes precipitated when exposed to low temperatures you may incubate it at 37 C for 15 20 min to resolubilize it 2 When do I need to use ProMelt and ProSink ProMelt Item 1115 is not needed if you will not recover the proteins ProSink Item 9030 is a protein precipitating reagent and it is required for blood DNA RNA extraction 3 How should I thaw 1 ml frozen blood in a 1 5 ml tube Ideally the
5. AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction AquaPreserve Instruction Manual General Information Description AquaPreserve is a multifunctional reagent for DNA RNA protein preservation and extraction It may be used to streamline biospecimen collection stabilization transport storage distribution and DNA RNA protein extraction By streamlining the entire biospecimen workflow AquaPreserve can reduce pre analytical variability increase data reproducibility and reliability AquaPreserve extracts total DNA RNA proteins from whole blood it recovers both cellular and cell free circulating DNA RNA proteins in the whole blood therefore maximizing the scientific value and utilities of the biospecimens Specification Product Name AquaPreserve Kit Product 8001 8060 Size 8001 1 ml 8060 60 ml sufficient for 240 mini 30 midi 15 maxi preps Kit Contents 8001 1 ml AquaPreserve Solution User Manual 8060 60 ml AquaPreserve Solution User Manual MSDS Available at www aquaplasmid com Storage Store tightly capped at 22 C Vortex the reagent to mix well before dispensing Note In addition to AquaPreserve please order ProSink 9030 for blood DNA and RNA extraction and ProMelt 1115 for protein extraction Terms amp Condition Product Usage For In Vitro Laboratory Research Use Only NOT to be administered to humans or used for medical d
6. fresh blood sample is aliquoted in tubes 3x of the blood sample volume or pre mixed with AquaPreserve and ProSink prior to freezing However to process existing 1 ml frozen blood sample in a 1 5 ml tube you may either cut open the tube to retrieve the frozen blood pellet or use 0 4 ml of AquaPreserve to partially thaw the frozen blood repeatedly and transfer it to a large tube 4 Why didn t I see the 28S and 18S rRNA bands in the gel The 28S and 18S rRNA bands may migrate with the genomic DNA in a native 0 8 agarose gel If you add some salts e g 30 uM NaOAc pH unadjusted to the loading dye you may get a better separation of the DNA and RNA bands However it would be better to do a DNase I digestion to remove the DNA before running the gel 5 How should I remove the genomic DNA from the DNA RNA preparation You may add 0 2 U of DNase I to 10 20 ul of DNA RNA solution in 0 5 1x DNase buffer and incubate at 22 37 C for 20 30 min Then run the digested sample in a 0 8 native agarose gel to confirm that the DNA digestion is complete To inactivate the DNase I you may use Ambion s DNase removal reagent or inactivate the DNase I at 65 C for 15 min 6 Can I do RT PCR without removing the contaminating genomic DNA Complete DNA removal may be difficult or unnecessary if you use intron spanning primers for the PCR amplification You may also design and use a 5 tailed RT primer to make the cDNA and then use a pair of PCR primers
7. h ProSink 1 Lyse the blood cells Add 0 25 ml of AquaPreserve to 0 25 ml of fresh or frozen whole blood in a 1 5 ml microfuge tube Shake and vortex to mix well and incubate at 22 C for 15 min Centrifuge at 14 000 xg for 5 min 2 Pellet the proteins Add 0 125 ml of ProSink to the centrifuged sample Shake and vortex to mix well Incubate at 22 C for 30 min Centrifuge at 14 000 xg for 5 min to pellet the proteins 3 Solubilize the proteins Remove the supernatant for DNA RNA precipitation Add 1 ml of ProMelt 1115 order separately to the wet protein pellet Pipet up and down to suspend the protein pellet Take 10 ul of the protein suspension and mix with 90 ul of ProMelt Vortex and incubate at 37 C for 10 min to solubilize the proteins completely for SDS PAGE B Protein precipitation with acetone 1 Lyse the blood cells Add 0 1 ml of AquaPreserve to 0 1 ml of fresh or frozen whole blood in a 1 5 ml microfuge tube Vortex or shake to mix well 2 Pellet the DNA RNA Add 0 7 vol 0 14 ml of isopropanol vortex for 60 sec and centrifuge at 14 000 xg for 5 min to pellet the blood DNA RNA 3 Pellet the proteins Transfer the protein containing supernatant 0 3 ml to a new 1 5 ml microfuge tube Add 4 vol 1 2 ml of acetone vortex for 60 sec and centrifuge at 14 000 xg for 5 min to pellet the proteins 4 Solubilize the proteins Decant to discard the supernatant tap the tube on a clean paper towel to remove residual acet
8. iagnosis Limited Product Warranty We offer a LIMITED PRODUCT WARRANTY to our customers This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by MultiTarget Pharmaceuticals We shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product Product Warning Contains guanidine thiocyanate Harmful if swallowed Causes irritation to skin eyes and respiratory tract Do not mix with Bleach Patents Trademarks amp Copyrights AquaPreserve is a trademark of MultiTarget Pharmaceuticals LLC 2015 Multitarget Pharmaceuticals LLC All rights reserved MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction AquaPreserve Blood DNA RNA Extraction Protocol This protocol uses 0 25 ml AquaPreserve 8060 and 0 125 ml ProSink 9030 ordered separately to extract DNA 12 ug and RNA 250 ng from 0 25 ml fresh or frozen human blood collected in regular anticoagulants 1 Lyse the blood cells Add 0 25 ml of AquaPreserve to 0 25 ml of fresh or frozen whole blood in a 1 5 ml microfuge tube Vortex and
9. ical variability Furthermore AquaPreserve extracts both cellular and cell free circulating DNA RNA from either fresh or frozen whole blood samples maximizing the value and utilities of the blood specimens 1 Stabilize the blood sample To stabilize the blood samples immediately transfer an aliquot of the blood sample e g 0 25 0 5 2 or 4 ml to a tube containing an equal volume of AquaPreserve Vortex to mix well Incubate the AquaPreserve lysed blood sample at room temperature for 30 min to 18 hrs Centrifuge at 14 000 xg for 5min Add 0 5 volume of ProSink e g for 1 ml AquaPreserve use 0 5 ml ProSink to the tube shake or vortex the tube to mix 2 Transport the blood sample The AquaPreserve and ProSink stabilized blood samples may be shipped at ambient temperature However to avoid exposing the samples to extreme heat in the summer days it is preferable to ship them on cold gel packs If it is not possible to stabilize the blood samples with AquaPreserve ProSink at collection they should be shipped on wet ice or dry ice by overnight carrier If the blood samples are shipped on dry ice it is preferable to aliquot the blood samples into tubes that can hold 3x of the blood sample volume so that AquaPreserve and ProSink may be added directly to the frozen blood samples later 3 Store the blood sample AquaPreserve and ProSink stabilized blood samples may be stored at 22 C for 1 5 days or at 4 C for 1 2 weeks For long term storage
10. o discard the alcohol solution Make sure the DNA RNA pellet remains in place before pouring off the alcohol solution Repeat the isopropanol rinse once Tap the tube on a paper towel to remove residual liquid and leave it upside down to air dry the DNA RNA pellet for 5 10 min 5 Solubilize the DNA RNA pellet Add 100 ul of deionized water to the DNA RNA pellet Vortex and or pipet to solubilize the DNA RNA Incubate at 22 C for 10 min Centrifuge again to pellet any insoluble and transfer the clear DNA RNA solution to a new tube Store at 20 C Table 1 Use the volume ratio of 1 1 0 5 blood AquaPreserve ProSink for other extraction scales Micro Mini Midi Maxi Blood ul 50 250 2 000 4 000 AquaPreserve ul 50 250 2 000 4 000 ProSink ul 25 125 1 000 2 000 Centrifuge tubes 0 6 ml 1 5 ml 15 ml 15 ml DNA yield ug 2 3 12 15 100 130 200 250 RNA yield ng 50 250 2 000 4 000 Number of extractions 1 200 240 30 15 MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction AquaPreserve for total blood DNA RNA biobanking AquaPreserve combines blood DNA RNA preservation with extraction It may be used to streamline blood collection stabilizaton transport storage distribution and DNA RNA extraction and reduce specimen pre analyt
11. one Immediately add 0 5 ml of ProMelt to the wet protein pellet pipette and vortex to suspend the pellet Incubate at 22 C for 15 min to solubilize the proteins Vortex and centrifuge at 14 000 xg for 5 min to pellet any insoluble Transfer the protein solution to a new microfuge tube and store at 4 or 20 C Some SDS may precipitate out at low temperatures however it will not interfere with SDS PAGE Alternatively it may be re solubilized by incubating at 65 C for 10 min MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction AquaPreserve Buffy Coat DNA RNA Protein Extraction Protocol If you need to recover the plasma for other assays or extract DNA RNA proteins from a large volume of blood you may prepare buffy coat from whole blood for DNA RNA protein extraction to reduce the consumption of the extraction reagents The protocol below is for processing 2 ml of whole blood to obtain 200 ul of buffy coat If you need to process larger volume of whole blood in the original vacutainer 5 10 ml you will simply scale up the reagent volumes proportionally 1 Prepare the buffy coat Centrifuge 2 ml of anticoagulated whole blood at 300 xg for 10 min at room temperature Remove some plasma 0 6 0 7 ml without disturbing the buffy coat Set the pipette at 100 1 and carefully
12. suck up the grayish buffy coat while slowly moving the tip across the interface and taking up as little RBC as possible Transfer the buffy coat to a 1 5 ml microfuge tube Repeat it by taking 100 ul of plasma just above the interface The total volume of buffy coat recovered is about 1 10 of the blood volume that is 200 pl 2 Lyse the blood cells Add one volume 200 ul of AquaPreserve to the buffy coat Vortex to mix well 3 Recover the DNA RNA Add 0 8 volume 320 ul of isopropanol to the cell lysate Vortex to mix well Centrifuge at 12 000 xg for 5 min at room temperature to pellet the DNA RNA Transfer 0 4 ml protein containing supernatant to a 2 ml tube for protein recovery Remove the remaining supernatant from the DNA RNA pellet as much as possible Fill up the microfuge tube with 50 isopropanol and quickly decant to discard the isopropanol solution Repeat the isopropanol rinse once Tap the tube on a clear paper towel to remove residual isopropanol and leave the tube up side down to air dry the DNA RNA pellet for 5 10 min Add 100 ul of deionized water to the pellet and vortex to suspend the DNA RNA pellet Incubate at room temperature for 10 min and centrifuge again to pellet any insoluble Transfer the DNA RNA solution to a new tube and store at 20 C 4 Recover the proteins Add 4 volumes 1 6 ml of acetone to the isopropanol supernatant obtained after DNA RNA precipitation Shake or vortex to mix well Centrifuge at 12 00
13. to mix well Incubate at 22 C for gt 30 min and then centrifuge to pellet the proteins save the pellet for protein recovery if desired and recover the clear lysate for DNA RNA precipitation with 1 volume of isopropanol MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaPreserve an aqueous solution for biospecimen preservation and biomolecule extraction AquaPreserve Blood Protein Extraction Protocol Total blood proteins may be extracted from fresh or frozen whole blood sample using one of the following two protocols The first protocol Protein precipitation with ProSink recovers blood proteins from ProSink precipitated protein pellet left over from blood DNA RNA extraction The second protocol Protein precipitation with acetone recovers blood proteins from AquaPreserve lysed blood directly by acetone precipitation independent of blood DNA RNA extraction The first protocol is convenient and streamlines blood DNA RNA protein extraction However the solubility of the proteins is lowered after being treated with ProSink If the concentration of your target protein is too low to be detected with the first protocol you may use the second protocol for protein precipitation to ensure all the proteins can be solubilized You may further increase the cellular protein concentration by extracting the proteins from the buffy coat A Protein precipitation wit
Download Pdf Manuals
Related Search