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=== Qubit dsDNA HS assay results === * Note that sample #2-P-Nu and #2-S-Nu were switched, so I will make changes to the data in the table later. <u>Raw data before normalization</u> : Sample #1 :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample Con.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Eluted volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' |- | #1-P-Q||<.5ng/mL||||||||NA||||24||NA |- | #1-P-Nu||<.5ng/mL||||||||NA||||24||NA |- | #1-P-CS||<.5ng/mL||||||||NA||||23||NA |- | #1-P-Z||<.5ng/mL||||||||Na||||33||NA |- | #1-S-Q||2.59||ng/mL||2||100||0.26||ug/mL||24.00||6.22 |- | #1-S-Nu||2.23||ng/mL||2||100||0.22||ug/mL||24.00||5.35 |- | #1-S-CS||3.78||ng/mL||2||100||0.38||ug/mL||23.00||8.69 |- | #1-S-Z||<.5ng/mL||||||||NA||||33.00||NA |- | #1-BC-Q||530||ng/mL||1||200||110.00||ug/mL||24.00||2,640.00 |- | #1-BC-Nu||67.7||ng/mL||2||100||6.77||ug/mL||24.00||162.48 |- | #1-BC-CS||12.2||ng/mL||2||100||1.22||ug/mL||23.00||28.06 |- | #1-BC-Z||94||ng/mL||2||100||9.40||ug/mL||33.00||310.20 |} : Sample #2 :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample Conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Eluted volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' |- | #2-P-Q||<.5ng/mL||||||||NA||||24.00||NA |- | #2-P-Nu||14.8||ng/mL||2||100||1.48||ug/mL||24.00||bgcolor="lawngreen"|35.52** |- | #2-P-CS||0.85||ng/mL||2||100||0.09||ug/mL||23.00||1.96 |- | #2-P-Z||<.5ng/mL||||||||NA||||33.00||NA |- | #2-S-Q||15.7||ng/mL||2||100||1.57||ug/mL||24.00||37.68 |- | #2-S-Nu||0.52||ng/mL||2||100||0.05||ug/mL||24.00||bgcolor="lawngreen"|1.25** |- | #2-S-CS||18.1||ng/mL||2||100||1.81||ug/mL||23.00||41.63 |- | #2-S-Z||3.58||ng/mL||2||100||0.36||ug/mL||33.00||11.81 |- | #2-BC-Q||143||ng/mL||1 of 10x dilution||2000||286.00||ug/mL||24.00||6,864.00 |- | #2-BC-Nu||474||ng/mL||2||100||47.40||ug/mL||24.00||1,137.60 |- | #2-BC-CS||19.5||ng/mL||1||200||3.90||ug/mL||23.00||89.70 |- | #2-BC-Z||102||ng/mL||1||200||20.40||ug/mL||33.00||673.20 |} : ** I suspected these two samples were switched maybe during sample aliquot or when I put the label to the tubes. <u>Normalized to 100ul sample input</u> : Sample #1 :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume used (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 100ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Rank for each<br>blood fraction''' |- | #1-P-Q||NA||100||NA|| |- | #1-P-Nu||NA||100||NA|| |- | #1-P-CS||NA||50||NA|| |- | #1-P-Z||NA||50||NA|| |- |bgcolor="cyan"| #1-S-Q||6.22||200||3.11||bgcolor="cyan"|2 |- |bgcolor="cyan"| #1-S-Nu||5.35||200||2.68||bgcolor="cyan"|3 |- |bgcolor="cyan"| #1-S-CS||8.69||100||8.69||bgcolor="cyan"|1 |- |bgcolor="cyan"| #1-S-Z||NA||50||NA||bgcolor="cyan"| |- | #1-BC-Q||2640.00||100||2,640.00||1 |- | #1-BC-Nu||162.48||100||162.48||3 |- | #1-BC-CS||28.06||50||56.12||4 |- | #1-BC-Z||310.20||50||620.40||2 |} : Sample #2 :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume used (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 100ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Rank for each<br>blood fraction''' |- | #2-P-Q||NA||150||NA|| |- | #2-P-Nu||35.52||200||bgcolor="lawngreen"|17.76||1 |- | #2-P-CS||1.96||50||3.91||2 |- | #2-P-Z||NA||50||NA|| |- | bgcolor="cyan"| #2-S-Q||37.68||200||18.84||bgcolor="cyan"|3 |- | bgcolor="cyan"| #2-S-Nu||1.25||150||bgcolor="lawngreen"|0.83||bgcolor="cyan"|4 |- | bgcolor="cyan"| #2-S-CS||41.63||100||41.63||bgcolor="cyan"|1 |- |bgcolor="cyan"| #2-S-Z||11.81||50||23.63||bgcolor="cyan"|2 |- | #2-BC-Q||6864.00||100||6,864.00||1 |- | #2-BC-Nu||1137.60||100||1,137.60||3 |- | #2-BC-CS||89.70||50||179.40||4 |- | #2-BC-Z||673.20||50||1,346.40||2 |} : <u>From the normalized tables above,</u> : '''Plasma DNA extraction''' :: - Sample #1 could not provide information since all DNA extracted from plasma were too low to be detectable by Qubit dsDNA HS :: - Sample #2 showed that NucleoSpin® Plasma XS kit had higher efficiency than ChargeSwitch® gDNA 1 ml Serum kit. However this comparison came from only one sample : '''Serum DNA extraction''' :: - Data from sample #1 and #2 showed that ChargeSwitch® gDNA 1 ml Serum kit had highest efficiency. :: - NucleoSpin® Plasma XS kit was the worst one for serum DNA extraction :: - ZymoBead™ Genomic DNA kit was better than QIAamp DNA Blood Mini Kit in sample #2 (close normalized yield), but had too low yield in sample #1 : '''Buffy coat DNA extraction''' :: - QIAamp DNA Blood Mini Kit had highest efficiency for buffy coat DNA extraction -> 2) ZymoBead™ Genomic DNA kit -> 3) NucleoSpin® Plasma XS kit -> 4) ChargeSwitch® gDNA 1 ml Serum kit :: - The results from sample #1 and #2 were consistent : - From this experiment, it looks like each kit had different efficiency in DNA extraction of different blood fractions. To get reliable result, I may need to include more samples for comparison if possible (seem like unlikely to have more samples to try). The choice of kit depends on what fraction of blood we are going to use to study. : - Another point that might affect the result of this experiment is the elution volume. For example, I used lower volume of elution volume for QIAGEN kit (26ul instead of 200ul) and ChargeSwitch kit (25ul instead of 50ul) because I wanted to minimize elution volume since bisulfite conversion need to fit volume of DNA in 20ul if I used Lightning kit (ready to use conversion reagent). This might cause poor DNA elution, especially the ChargeSwitch kit. : - This result apparently showed that we can get more DNA from serum fraction than plasma ---- : - I will continue treating extracted DNA with sodium bisulfite to compare the yield with the experiment that bisulfite conversion was applied after DNA binding to beads (on samples with minimum 10ng in 100ul sample).
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