BDNF Val66Met Polymorphism, the Allele-Specific Analysis by QRT-PCR – a Novel Protocol – Aprofarm Formulación Magistral Farmacológica

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by QRT-PCR – a Novel Protocol

Molecular Blood Group Screening in Donors from Arabian Countries and Iran Using High-Throughput MALDI-TOF Mass Spectrometry and PCR-SSP

Background and aims: Only little is known about blood groups other than ABO blood groups and Rhesus factors in Arabian countries and Iran. During the last years, increased migration to Central Europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. Therefore, blood group allele frequencies should be determined in individuals from Arabian countries and Iran by molecular typing and compared to a German rare donor panel.

Methods: 1,111 samples including 800 individuals from Syria, 147 from Iran, 123 from the Arabian Peninsula, and 41 from Northern African countries were included in a MALDI-TOF MS assay to detect polymorphisms coding for Kk, Fy(a/b), Fynull, Cw, Jk(a/b), Jo(a+/a-), Lu(a/b), Lu(8/14), Ss, Do(a/b), Co(a/b), In(a/b), Js(a/b), Kp(a/b), and variant alleles RHCE*c.697C>G and RHCE *c.733C>G. Yt(a/b), S-s-U-, Velnull, Conull, and RHCE *c.667G>T were tested by PCR-SSP.

Results:  Of the Arabian donors, 2% were homozygous for the FY *02.01N allele (Fynull), and 15.7% carried the heterozygous mutation. However, 0.8% of the German donors also carried 1 copy of the allele. 3.6% of all and 29.3% of Northern African donors were heterozygous for the RHCE *c.733C>G substitution, 0.4% of the Syrian probands were heterozygous for DO *01/DO *01.-05, a genotype that was lacking in German donors.

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Whereas the KEL *02.06 allele coding for the Js(a) phenotype was missing in Germans; 0.8% of the Syrian donors carried 1 copy of this allele. 1.8% of the Syrian but only 0.3% of the German donors were negative for YT *01. One donor from Northern Africa homo-zygously carried the GYPB *270+5g>t mutation, inducing the S-s-U+w phenotype, and in 2 German donors a GYPB *c.161G>A exchange, which induces the Mit+ phenotype, caused a GYPB *03 allele dropout in the MALDI assay. The overall failure rate of the Arabian panel was 0.4%.

CRISPR-mediated knock-in in the mouse embryo using long single stranded DNA donors synthesised by biotinylated PCR

Successful gene knock-in by CRISPR-Cas9 in the mouse zygote requires three components; guideRNA, Cas9 protein and a suitable donor template, which usually comprises homology flanked insert sequence. Recently, long single stranded DNA (lssDNA) donors have emerged as a popular choice of DNA donor, outperforming dsDNA templates in terms of knock-in efficiency for gene tagging and generating conditional alleles.

The generation of these donors can be achieved through several methods that may introduce errors in the sequence, result in poor yields, and contain dsDNA contamination. We have developed our own cost-effective lssDNA synthesis methodology that results in high purity, sequence verified, low contamination lssDNA donors. We provide a detailed methodology on the design and generation of such donors for gene tagging experiments and generating conditional alleles.

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR – a Novel Protocol

Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene (BDNF). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning.

Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF.

Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) – relative to housekeeping genes – and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR).

Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion: Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism.

Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized.

This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.

Droplet Digital PCR Analysis of Liquid Biopsy Samples Unveils the Diagnostic Role of hsa-miR-133a-3p and hsa-miR-375-3p in Oral Cancer

Despite the availability of screening programs, oral cancer deaths are increasing due to the lack of diagnostic biomarkers leading to late diagnosis and a poor prognosis. Therefore, there is an urgent need to discover novel effective biomarkers for this tumor. On these bases, the aim of this study was to validate the diagnostic potential of microRNAs (miRNAs) through the analysis of liquid biopsy samples obtained from ten oral cancer patients and ten healthy controls. The expression of four selected miRNAs was evaluated by using droplet digital PCR (ddPCR) in a pilot cohort of ten oral cancer patients and ten healthy donors. Bioinformatics analyses were performed to assess the functional role of these miRNAs.

The expression levels of the predicted down-regulated hsa-miR-133a-3p and hsa-miR-375-3p were significantly reduced in oral cancer patients compared to normal individuals while no significant results were obtained for the up-regulated hsa-miR-503-5p and hsa-miR-196a-5p. ROC analysis confirmed the high sensitivity and specificity of hsa-miR-375-3p and hsa-miR-133a-3p. Therefore, both miRNAs are significantly down-regulated in cancer patients and can be used as biomarkers for the early diagnosis of oral cancer. The analysis of circulating miRNAs in a larger series of patients is mandatory to confirm the results obtained in this pilot study.

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Description: 2X, High ROX, 5ml

Apex qPCR GREEN Master Mix
42-121PG	Genesee Scientific	4000 x 25µl Reactions/Unit	EUR 3095.57
Description: 2X, High ROX, 50ml

BioEasy Master Mix (SYBR Green, No ROX)
TRI-B25L1D	TRI Biotech	200T	EUR 403.2

BioEasy Master Mix (SYBR Green, low ROX)
TRI-B25L1C	TRI Biotech	200T	EUR 403.2

BioEasy Master Mix (SYBR Green, high ROX)
TRI-B25L1B	TRI Biotech	200T	EUR 403.2

ChamQ SYBR Color qPCR Master Mix
Q411-00-100rxns	Vazyme	100 rxns	EUR 1.09

ChamQ SYBR Color qPCR Master Mix
Q411-02	Vazyme	500 rxns (20 μl/rxn)	EUR 140.63

ChamQ SYBR Color qPCR Master Mix
Q411-02-500rxns	Vazyme	500 rxns	EUR 145.62

ChamQ SYBR Color qPCR Master Mix
Q411-03	Vazyme	2,500 rxns (20 μl/rxn)	EUR 634

ChamQ SYBR Color qPCR Master Mix
Q411-03-2500rxns	Vazyme	2,500 rxns	EUR 656.51

AceQ Universal SYBR qPCR Master Mix
Q511-00-100rxns	Vazyme	100 rxns	EUR 1.09

AceQ Universal SYBR qPCR Master Mix
Q511-02	Vazyme	500 rxn (20 μl/rxn)	EUR 152.72

AceQ Universal SYBR qPCR Master Mix
Q511-02-500rxns	Vazyme	500 rxns	EUR 158.14

AceQ Universal SYBR qPCR Master Mix
Q511-03	Vazyme	2500 rxn (20 μl/rxn)	EUR 688.5

AceQ Universal SYBR qPCR Master Mix
Q511-03-2500rxns	Vazyme	2,500 rxns	EUR 712.94

ChamQ Universal SYBR qPCR Master Mix
Q711-00-100rxns	Vazyme	100 rxns	EUR 1.09

ChamQ Universal SYBR qPCR Master Mix
Q711-02	Vazyme	500 rxn (20 μl/rxn)	EUR 152.72

ChamQ Universal SYBR qPCR Master Mix
Q711-02-500rxns	Vazyme	500 rxns	EUR 158.14

ChamQ Universal SYBR qPCR Master Mix
Q711-03	Vazyme	2500 rxn (20 μl/rxn)	EUR 688.5

ChamQ Universal SYBR qPCR Master Mix
Q711-03-2500rxns	Vazyme	2,500 rxns	EUR 712.94

miRNA Universal SYBR qPCR Master Mix
MQ101-00-100rxns	Vazyme	100 rxns	EUR 1.09

miRNA Universal SYBR qPCR Master Mix
MQ101-01	Vazyme	125 rxn(20 μl/rxn)	EUR 38.9

miRNA Universal SYBR qPCR Master Mix
MQ101-01-125rxns	Vazyme	125 rxns	EUR 40.28

miRNA Universal SYBR qPCR Master Mix
MQ101-02	Vazyme	500 rxn(20 μl/rxn)	EUR 145

miRNA Universal SYBR qPCR Master Mix
MQ101-02-500rxns	Vazyme	500 rxns	EUR 150.15

HotStart™ 2X Green qPCR Master Mix
K1070-25	ApexBio	25ml	EUR 392

Description: Reverse Transcription & PCR\|qPCR

HotStart™ 2X Green qPCR Master Mix
K1070-5	ApexBio	5ml	EUR 124

Description: Reverse Transcription & PCR\|qPCR

HotStart™ 2X Green qPCR Master Mix
K1070-50	ApexBio	50ml	EUR 768

Description: Reverse Transcription & PCR\|qPCR

2×SYBR Green qPCR Mix
E37F021-10x5mL	EnoGene	10×5mL	EUR 1360
Description: DsDNA, ssDNA, RNA quantitative detection kit

2×SYBR Green qPCR Mix
E37F021-5mL	EnoGene	5mL	EUR 227
Description: Fluorescent quantitative PCR kits

2×SYBR Green qPCR Mix
MBS8579840-200Tests	MyBiosource	200Tests	EUR 285

2×SYBR Green qPCR Mix
MBS8579840-50Tests	MyBiosource	50Tests	EUR 180

2×SYBR Green qPCR Mix
MBS8579840-5x200Tests	MyBiosource	5x200Tests	EUR 1230

2×SYBR Green qPCR Mix
MBS8579840-5x50Tests	MyBiosource	5x50Tests	EUR 770

ChamQ SYBR qPCR Master Mix (Without ROX)
Q321-02	Vazyme	500 rxns (20 μl/rxn)	EUR 120.55

ChamQ SYBR qPCR Master Mix (Without ROX)
Q321-02-500rxns	Vazyme	500 rxns	EUR 124.83

ChamQ SYBR qPCR Master Mix (Without ROX)
Q321-03	Vazyme	2,500 rxns (20 μl/rxn)	EUR 543.5

ChamQ SYBR qPCR Master Mix (Without ROX)
Q321-03-2500rxns	Vazyme	2,500 rxns	EUR 562.79

Taq Pro Universal SYBR qPCR Master Mix
Q712-00-100rxns	Vazyme	100 rxns	EUR 1.09

Taq Pro Universal SYBR qPCR Master Mix
Q712-02	Vazyme	500 rxns (20 μl/rxn)	EUR 161

Taq Pro Universal SYBR qPCR Master Mix
Q712-02-500rxns	Vazyme	500 rxns	EUR 166.72

Taq Pro Universal SYBR qPCR Master Mix
Q712-03	Vazyme	2,500 rxns (20 μl/rxn)	EUR 725

Taq Pro Universal SYBR qPCR Master Mix
Q712-03-2500rxns	Vazyme	2,500 rxns	EUR 750.74

miRNA SYBR Green qPCR Mix
4308A	Unibiotest	100 Tests*20μL	EUR 80

miRNA SYBR Green qPCR Mix
4308B	Unibiotest	200 Tests*20μL	EUR 150

miRNA SYBR Green qPCR Mix
4308C	Unibiotest	500 Tests*20μL	EUR 300

ViPrimePLUSHigh 5X One Step RT-qPCR Green Master Mix I (SYBR® Green Dye), 100rxns
QLMM10	Vivantis	each	Ask for price

HotStart™ 2X FAST Green qPCR Master Mix
K1171-25	ApexBio	25ml	EUR 424
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

HotStart™ 2X FAST Green qPCR Master Mix
K1171-5	ApexBio	5ml	EUR 132
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

HotStart™ 2X FAST Green qPCR Master Mix
K1171-50	ApexBio	50ml	EUR 826.4
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

HotStart™ Universal 2X Green qPCR Master Mix
K1170-25	ApexBio	25ml	EUR 392
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

HotStart™ Universal 2X Green qPCR Master Mix
K1170-5	ApexBio	5ml	EUR 124
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

HotStart™ Universal 2X Green qPCR Master Mix
K1170-50	ApexBio	50ml	EUR 752
Description: Reverse Transcription & PCR\|qPCR/Real-time PCR

ChamQ SYBR qPCR Master Mix (Low ROX Premixed)
Q331-02	Vazyme	500 rxns (20 μl/rxn)	EUR 120.55

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BDNF Val66Met Polymorphism, the Allele-Specific Analysis by QRT-PCR – a Novel Protocol

Molecular Blood Group Screening in Donors from Arabian Countries and Iran Using High-Throughput MALDI-TOF Mass Spectrometry and PCR-SSP

Benzidines Mix (2000ppm) - 5.5mL Bottle - 5.5ML

8260 ISTD Mix (15mL bottle) - 15ML

BRAND(TM) GLASS BOTTLE-TOP TILT PIPETTE

BRAND(TM) GLASS BOTTLE-TOP TILT PIPETTE

BRAND(TM) GLASS BOTTLE-TOP TILT PIPETTE

BRAND(TM) GLASS BOTTLE-TOP TILT PIPETTE

Acid Spiking Mix (2000ppm) - 5.5mL Bottle - 5.5ML

Semivolatile ISTD Mix (4000ppm) - 5.5mL Bottle - 5.5ML

Semivolatile TCLP Mix (2000ppm) - 5.5mL Bottle - 5.5ML

BRAND(TM) GLASS BOTTLE-TOP TILT PIPETTE - EACH

Base/Neutral Spiking Mix (1000ppm) - 5.5mL Bottle - 5.5ML

Semivolatile ISTD Mix (6) (2000ppm) - 5.5mL Bottle - 5.5ML

EPA Method 625 Base/Neutral Mix 1 (2000ppm) - 5.5mL Bottle - 5.5ML

EtBr Dropper Bottle, 10ml

Base/Neutral Surrogate Mix (3) (1000ppm) - 5.5mL Bottle - 5.5ML

Base/Neutral Surrogate Mix (4) (1000ppm) - 5.5mL Bottle - 5.5ML

Base/Neutral Surrogate Mix (3) (1000ppm) - 11.5mL Bottle - 11.5ML

Semivolatile Surrogate Mix (6) (1000/2000ppm) - 5.5mL Bottle - 5.5ML

Acid Extractables Surrogate Mix (3) (2000ppm) - 5.5mL Bottle - 5.5ML

Acid Extractables Surrogate Mix (4) (2000ppm) - 5.5mL Bottle - 5.5ML

Acid Extractables Surrogate Mix (3) (2000ppm) - 11.5mL Bottle - 11.5ML

Ethidium Bromide Dropper Bottle

Ethidium Bromide Dropper Bottle

Bottle Dropping 30ml Glass - PK6

Bottle Bijou Hw 7ml No Cap - PK255

Bottle Duran Amber 1L No Cap - EACH

Bottle Duran Amber 2L No Cap - PK10

Base/Neutral Surrogate Mix 4 component-10.5mL Bottle - 10.5ML

Bottle Alpha amber 125ml no cap - PK104

Nalgene Bottle Dropper 4ml LDPE Clear

Nalgene Bottle Dropper 8ml LDPE Clear

Nalgene Bottle Dropper 15ml LDPE Clear

Nalgene Bottle Dropper 15ml LDPE White

Acid Extractables Surrogate Mix 4 component-10.5mL Bottle - 10.5ML

Bottle Dropping 50ml Glass TK Pattern - EACH

Nalgene Bottle Dropper 4ml LDPE Clear - PK25

Nalgene Bottle Dropper 8ml LDPE Clear - PK25

Nalgene Bottle Dropper 4ml LDPE White - PK25

Nalgene Bottle Dropper 8ml LDPE White - PK25

Ethidium bromide dropper bottle 10 mL - EACH

Nalgene Bottle Dropper 15ml LDPE Clear - PK25

Nalgene Bottle Dropper 15ml LDPE White - PK25

Bottle 60ml Amber Bottle - PK12

Duran Bottle 100ml Square Clear

Duran Bottle 100ml Square Amber

Duran Bottle 250ml Square Clear

Duran Bottle 250ml Square Amber

Duran Bottle 500ml Square Clear

Duran Bottle 500ml Square Amber

Wash bottle assembly (1 bottle with cap and tubing)

Duran Bottle 1000ml Square Amber - PK10

6oz Square Bottom Bottle (PP)

6oz Square Bottom Bottle (PE)

Bottle 1L HDPE Square - PK5

Nalgene Bottle 500ml PP Square - PK12

Nalgene Bottle 125ml PE Square - PK12

Nalgene Bottle 250ml PE Square - PK12

Nalgene Bottle 500ml PE Square - PK12

Duran Bottle 100ml Square Clear - PK10

Duran Bottle 100ml Square Amber - PK10

Duran Bottle 250ml Square Clear - PK10

Duran Bottle 250ml Square Amber - PK10

Duran Bottle 500ml Square Clear - PK10

Duran Bottle 500ml Square Amber - PK10

Kartell Bottle 250ml HDPE Square - PK10

Kartell Bottle 500ml HDPE Square - PK10

Duran Bottle 1L Square Clear

Pyrex Reagent Bottle 500ml Square - PK10

Bottle Nalge Square 250Ml - PK72

Bottle 50ml Square Clear - PK10

Duran Laboratory Bottle with GL45 neck no Cap 500ml - EACH

Nalgene Bottle 250ml PP Square WM - PK12

Nalgene Bottle 500ml PP Square WM - PK12

Bottle 150ml hdpe Square Red cap - PK300

Nalgene Bottle 125ml PC Square Media - PK6

Nalgene Bottle 250ml PC Square Media - PK6

Nalgene Bottle 500ml PC Square Media - PK4

Nalgene Bottle 1L PP Square - PK6