NucleoSpin® Plasma XS NucleoSpin® Plasma XS XS columns – new column design for specific applications New column design – why in general? - customer do more and more often start with very small samples (e.g. for expression profiling, single cell characterizations) - customer asked for: high recovery of RNA/DNA in very small elution volumes New column design – why for plasma? - DNA in plasma is of low concentration and highly fragmented, standard kits fail to give satisfying DNA recovery and concentration Standard columns – the problems: - dead volumes of standard mini kits are normally to high to allow an elution in less than 50µl - smaller columns would not fit in standard centrifuges What to do? NucleoSpin® Plasma XS Qiagen‘s solution – MinElute columns standard columns broader thrust ring, smaller menbrane diameter thin silica menbrane The result: elution in 20 - 50µl possible Customer still ask for less elution volume NucleoSpin® Plasma XS New columns – the design of our columns standard NucleoSpin columns funnel shaped thrust ring small membrane diameter small elution volumes Xtra Small elution volume Xtra Sensitive colums Xtra high DNA concentration NucleoSpin® Plasma XS Simple and fast procedure, in less than 30 min from plasma to circulating DNA NucleoSpin® Plasma XS Recovery of fragmented DNA Recovery rates from plasma spikes DNA spikes of 50, 100, 150, 250, and 1000 bp were mixed with a plasma sample. DNA was subsequently purified with NucleoSpin® Plasma XS and a kit of competitor Q in parallel. MACHEREY-NAGEL in-house data Competitor Q completely failed to show recovery for fragments smaller than 100 bp and resulted in an overall weaker performance compared to NucleoSpin® Plasma XS (MN) which yielded significantly higher DNA concentration. NucleoSpin® Plasma XS DNA yield with multiple column loadings 100 Five different plasma samples with a different total DNA content were used as sample for DNA purification. Multiples of the standard lysate (240µl plasma + 360µl binding buffer) were loaded. 10 Yield significantly increases at least Plasma 1 DNA yield [ng] over the first three loading steps, thus the kit can be highly recommended for Plasma 2 up to 740 µl of plasma, Plasma 3 MACHEREY-NAGEL in-house data Plasma 4 1 DNA yield increases from Plasma 5 one to three column loadings (for plasma of low as well as for plasma of high DNA 0,1 content) 0 1 2 3 4 Number of column loadings with 600 µl lysate 480µl plasma + 240µl plasma + 720µl plasma + 720µl binding buffer 360µl binding buffer 1080µl binding buffer NucleoSpin® Plasma XS Elution profile - concentration and yield DNA recovery depends on the elution volume. Below a certain volume, DNA yield decreases significantly with an decreasing elution volume (due to the dead volume of the silica membrane). Qiagen´s MinElute columns require at least 20µl elution volume. Below this, yield (blue) decreases and DNA concentration (red) can not be increased (Q). NucleoSpin XS columns allow a significantly smaller elution volume, thus DNA concentration (red) can significantly be NucleoSpin Plasma XS allows small increased (MN). elution volumes and results in highly MACHEREY-NAGEL in-house data concentrated DNA NucleoSpin® Plasma XS Sales arguments Arguments Customer benefits ► silica membrane technology …known for high reliability ► optimized column design …reduced membrane area ► elution in only 5 - 20 μl …gives you highly concentrated DNA ► high DNA concentration …no need for subsequent concentration ► high purity …ready-to-use for real-time PCR ► high recovery of fragments >50bp …ideal for fragmented circulating DNA ► fast preparation, easy handling … convenient in use, requires less than 30 min ► standard protocol for 240 μl plasma … ideal solution for your routine analysis ► multiple loading steps possible … a solution even for up to ~720 µl of plasma A solution for an existing problem, no other kit is existing in the market yet! NucleoSpin® Plasma XS Application data Analysis of cancer NucleoSpin® Plasma XS Analysis of cancer Biopsy analysis: In case of cancer suspicion (e.g. tissue alteration), a common diagnostic tool is the extraction of a biopsy sample for subsequent histological and genetic analysis Risks and Intricacies? - injury, bleedings - infections - vascular and organ damage - spreading of cancer cells None invasive alternatives requested! How can NucleoSpin® Plasma XS help? NucleoSpin® Plasma XS Analysis of cancer Increased amounts of circulating DNA in plasma have been found in disorders including cancer, autoimmune diseases, infections, certain fetal diseases, trauma, stroke, and others. Tumor cells Lymphocytes destroy tumor cells Degradation Cells affected products circulate Apoptosis deregulation in blood plasma by disorders Blood draw / Plasma recovery - oncogenes Circulating DNA: - tumor suppr. genes NucleoSpin® Plasma XS - highly fragmented - microsat. alterations - low in concentration - point mutations None invasive , highly sensitive NucleoSpin® Plasma XS Analysis of cancer NucleoSpin® Plasma XS is superior to competitor Midi kits - Better PCR signals from less volume of plasma Starting material: Plasma samples originating from rat EDTA blood PCR amplification: 165 bp fragment of the k-ras gene (often involved in cancer). Comparison: QIAamp DNA Blood Midi, NucleoSpin® Plasma XS QIAamp DNA Blood Midi, input 1000 μl plasma, purification according to the user manual, modification: 100μl elution buffer were pre-heated to 56°C, 5 min incubation on the membrane prior to elution, elution fraction concentrated to 30 μl, 2 μl of concentrated eluate used for PCR NucleoSpin® Plasma XS, input 240 μl plasma, purification according to the user manual without modification, NucleoSpin® Plasma XS shows an elution with 20 μl, 2 μl of eluate used earlier PCR signal of 2.1 cycles even for PCR though QIAamp DNA Blood Midi Data kindly provided by Dolores C. García- represents a 2.7 fold PCR input Olmo, PhD, Unidad de Investigación, Complejo Hospitalario Universitario de compared to NucleoSpin® Plasma XS. Albacete, Albacete, Spain NucleoSpin® Plasma XS Application data Analysis of fetal DNA NucleoSpin® Plasma XS Analysis of fetal DNA Today´s solution: Amniocentesis - amniotic fluid contains fetal cells for early DNA diagnosis When indicated? - pregnant women > 35 of age , higher risk of genetic mutations, risk of handicaps - genetic defects in the family - identification of developmental disorders - test of blood group incompatibility Risks and Intricacies? - injury of the child by injection needle - bleeding in the uterus - rate of abortion 0,5%! None invasive replacement of Amniocentesis requested! How can NucleoSpin® Plasma XS help? NucleoSpin® Plasma XS Analysis of fetal DNA Future´s Solution: Placental circulation connects maternal and fetal bloodstream Fetal and maternal circulations are separated by placental membranes. This barrier seems to be incomplete to cellular trafficking Molecular analysis of plasma DNA during human pregnancy has led to the discovery that maternal plasma contains both fetal and maternal DNA. Conclusions: Non invasive blood draw from pregnant woman can replace amniocentesis Robust and reliable nucleic acid purification kit required (especially for fragmented cell free DNA) NucleoSpin® Plasma XS NucleoSpin® Plasma XS Analysis of fetal DNA NucleoSpin® Plasma XS is ideally suited for the detection of fetal DNA in maternal plasma Starting material: Human maternal plasma used for fetal DNA diagnostics Duplex PCR amplification: RHD fragment (Rhesus-gene) and an Amelogenin fragment specific for the male Y-chromosome, amplicons 150 – 180 bp in size. Comparison: Roche MagNA Pure Compact Nucleic Isolation Kit I - Large Volume, NucleoSpin® Plasma XS. Roche MagNA Pure Compact Nucleic Isolation Kit I – Large Volume, input 1000 μl plasma, purification according to the user manual, 10% of the eluate used for PCR (i.e. DNA from approx. 100 μl plasma) NucleoSpin® Plasma XS, input 240 μl plasma, purification according to The significantly better performance of the user manual, 40% of the eluate used for PCR (i.e. DNA from approx. 100 μl NucleoSpin® Plasma XS is demonstrated by plasma) an earlier PCR signal of 2.6 cycles (RHD gene) and 2.1 cycles (Amelogenin) Data kindly provided by Dr. respectively. Doescher, DRK Blutspendedienst N.S.T.O.B.,Oldenburg, Germany NucleoSpin® Plasma XS Customer testimonals We tested NucleoSpin® Plasma XS using human maternal plasma samples for subsequent fetal blood group genotyping (fetal RHD, RHE, and KEL typing). NucleoSpin® Plasma XS gave a 2.5 – 3 cycles earlier signal compared to the competitor kit we used so far. I like the easy handling, the short purification procedure, and the good results! Dr. Doescher, DRK Blutspendedienst NSTOB, Oldenburg, Germany We tested NucleoSpin® Plasma XS using rat plasma samples and are very satisfied. In plasma samples from healthy rats, we found significantly higher amounts of DNA compared to the competitor kit we used so far (PCR target: k-ras). In addition to this, less volume of plasma is sufficient (240 μl in contrast to 1 ml which we needed with the present kit) and the procedure is quicker! Dolores C. García-Olmo, PhD, Unidad de Investigación, Complejo Hospitalario Universitario de Albacete, Albacete, Spain NucleoSpin® Plasma XS Summary NucleoSpin® Plasma XS The only kit in the market that gives you highly concentrated circulating DNA from plasma - higher yields and recovery rates than competitors! - less plasma needed - a mini prep is often enough for analysis - not available from Qiagen - not only an alternative… a solution!