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Research Article
This research discovered increased levels of HMGA1 and decreased levels of HAND1 expression in laryngeal cancer, indicating an inverse relationship and significant associations with metastasis and clinical stage, suggesting that both contribute to tumor progression.
This study investigated the expression profiles of the HMGA1 and HAND1 genes in laryngeal squamous cell carcinoma (LSCC) as well as in adjacent normal laryngeal mucosal tissues through the application of immunohistochemistry and RT-PCR methodologies. The objective was to investigate their possible roles in the initiation, advancement, invasion, and metastasis of LSCC. Both HMGA1 and HAND1 were detected in cancerous and adjacent normal tissues. Nevertheless, HMGA1 demonstrated a significantly elevated expression in LSCC tissues, while HAND1 displayed considerably reduced expression levels. Elevated HMGA1 expression was positively associated with lymph node metastasis (100.0% vs. 50.0%) and advanced clinical stage (91.7% vs. 44.4%), but was not associated with tumor histological grade, clinical type, or patient age. In contrast, reduced HAND1 protein expression was associated with lymph node metastasis, clinical stage, and pathological grade, suggesting a role in tumor progression and metastasis. Furthermore, a linear negative correlation was noted between the expressions of HMGA1 and HAND1, suggesting a potential regulatory interaction in which HMGA1 may suppress HAND1 expression. These results imply that HMGA1 and HAND1 have complementary functions in the pathogenesis of LSCC, with HMGA1 potentially facilitating tumor development and HAND1 serving as a suppressor. The interaction between these two genes may offer new perspectives on the molecular mechanisms that drive LSCC progression and metastasis.
Laryngeal cancer is the second most common primary malignant tumor of the head and neck, following nasopharyngeal cancer, accounting for 26%–30% of all head and neck tumors worldwide1. Over 90% of laryngeal cancers are classified as laryngeal squamous cell carcinoma (LSCC)2. The primary treatments for laryngeal cancer are surgery and radiotherapy. Local recurrence and lymph node metastasis are the leading causes of death in laryngeal cancer patients. Therefore, effective management of recurrence and metastasis remains a major clinical challenge. With the advancement of molecular biology, various genes with abnormal expression in laryngeal cancer have been identified. However, the mechanisms underlying tumor invasion and metastasis in laryngeal cancer remain unclear and remain an area requiring further investigation.
High mobility group A (HMGA) proteins are non-histone chromatin-binding proteins located in the cell nucleus. HMGA1 binds to DNA and associated proteins, inducing chromatin structural alterations that regulate gene transcription3. As a chromatin-associated transcriptional regulator, HMGA1 modulates the expression of multiple tumor-related genes involved in proliferation and metastasis. Previous studies have demonstrated that HMGA1 is overexpressed in a wide range of human cancers, including thyroid, colorectal, pancreatic, and head and neck malignancies4,5,6,7, as well as other epithelial and solid tumors reported in the literature. HMGA1 regulates tumorigenesis by modulating the expression of multiple downstream target genes involved in proliferation and metastasis8.
Heart and neural crest derivatives expressed (HAND) genes are members of the bHLH (basic helix–loop–helix) family, including HAND1 and HAND2. HAND1, as a transcription factor, plays a role in trophoblast differentiation and heart development9. However, the expression mechanism of HAND1 in the extra-embryonic cell layer remains unclear. It has been confirmed that HMGA1 directly binds to the HAND1 promoter, inhibiting HAND1 activity both in vivo and in vitro10. HAND1, as a repressor protein, has been implicated in regulating cell differentiation and cancer formation. Studies have found that the expression of HAND1 is silenced and hypermethylated in human gastric cancer11, pancreatic cancer12, ovarian cancer13, and thyroid cancer14. However, to our knowledge, limited studies have investigated HAND1 expression in laryngeal cancer tissues.
In the present study, immunohistochemistry (two-step detection method) and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HMGA1 and HAND1 proteins and mRNA in LSCC and adjacent normal mucosal tissues. Immunohistochemistry enables visualization of protein localization within tissue architecture, whereas RT-PCR allows sensitive detection of gene expression at the transcriptional level, providing complementary insights into protein and mRNA expression patterns. Compared with quantitative PCR (qPCR), which offers higher sensitivity and precise quantification, Western blotting, which enables protein-level validation but lacks spatial context, and RNA sequencing (RNA-seq), which provides comprehensive transcriptomic profiling but requires advanced computational analysis, the combined use of immunohistochemistry and RT-PCR represents a cost-effective and clinically applicable strategy for biomarker investigation. The study aimed to explore the role and correlation of these two factors in the occurrence, development, progression, and metastasis of LSCC. Furthermore, evaluating HMGA1 and HAND1 expression may provide biomarkers for diagnosis and prognostic assessment in laryngeal cancer, potentially improving risk stratification and aiding clinical decision-making.
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This study was reviewed and approved by the Research Ethics Committee of Shijiazhuang People’s Hospital (Approval No. YKLS 2026 No. 041; March 27, 2026). The study was conducted in accordance with the Regulations on Ethical Review of Biomedical Research Involving Human Subjects, the Regulations on Ethical Review of Life Science and Medical Research Involving Human Subjects, the Declaration of Helsinki, and the International Ethical Guidelines for Health-Related Research Involving Humans. Human tissue samples were obtained from archived surgical specimens in compliance with institutional ethical requirements, ensuring anonymization and confidentiality of patient data. Written informed consent was obtained from participants, or a waiver of consent was granted by the ethics committee for the use of anonymized archived specimens, as applicable.
Experimental materials
Source of specimens
A total of 42 cases of LSCC tissues and 25 cases of adjacent normal laryngeal mucosa tissues were obtained from surgical resection specimens of LSCC at the Otolaryngology-Head and Neck Surgery Department of Hebei between February 2011 and August 2012. Among these, 25 cases of adjacent normal mucosa (taken from a site > 1.0 cm from the cancer edge) were pathologically confirmed as inflammatory or normal mucosa. The 42 LSCC specimens were all pathologically confirmed as squamous cell carcinoma. All tissues were stored at -80 °C. The 42 patients were male, with no female patients. Among the LSCC cases, 15 were well-differentiated, 18 were moderately differentiated, and 9 were poorly differentiated. Eighteen cases had lymph node metastasis, while 24 cases did not. The clinical staging was based on the TNM classification and staging system (UICC, 2002), with 20 cases in stages I and II, and 22 cases in stages III and IV. There were 30 cases of supraglottic type, 11 cases of glottic type, and 1 case of subglottic type. The age range of patients was 40–75 years, with a median age of 60 years. None of the patients received radiotherapy or chemotherapy before surgery, and clinical data were complete.
Experimental methods and steps
HE staining procedure
Slide preparation and hematoxylin–Eosin staining
Glass slides for immunohistochemistry were first soaked in concentrated acid for 24 h, followed by thorough washing under running tap water for at least 10 min to remove residual acid, and immersion in distilled water for 30 min twice with gentle agitation to ensure complete rinsings. After soaking in 95% ethanol for 2 h, the slides were dried in a 60 °C oven, then immersed in an APES: acetone (1:49, v/v) mixture for 1 min with gentle shaking to ensure uniform coating, followed by rinsing with pure acetone. Finally, they were air-dried at room temperature (20–25 °C) to ensure proper tissue adhesion during subsequent staining procedures.
The paraffin-embedded tissue sections were sequentially immersed in xylene I and xylene II for 10 min each to remove paraffin, followed by rehydration through graded ethanol (100%, 95%, 80%) for 5–10 min each, and then rinsed in distilled water for 2 min. The sections were then subjected to hematoxylin–eosin (HE) staining as follows: hematoxylin staining for 4 min, rinsing under running tap water for 3 min with continuous flow to remove excess stain, differentiation using 1% hydrochloric acid in ethanol for 30 s, washing three times with tap water to terminate differentiation, and eosin staining for 10 s. Routine graded dehydration was performed sequentially by successively using 70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 5 min each. After drying, the slide was mounted using neutral balsam.
Immunohistochemistry procedure
Paraffin sections of 4 µm thickness were cut, laid flat on APES-coated slides, and placed in a 67 °C oven overnight, followed by deparaffinization with Xylene I and II for 10 min each. Gradual dehydration was carried out using 100%, 90%, and 70% alcohol for 5 min each, followed by washing with tap water and distilled water for 3 min. To block endogenous peroxidase activity, slides were incubated with freshly prepared 3% methanol-hydrogen peroxide solution, ensuring complete coverage of the tissue section at room temperature (20–25 °C) for 20 min, followed by three washes with distilled water for 5 min in 0.01 M PBS.
Antigen retrieval was performed by immersing slides in citrate buffer (pH 6.0) and heating in a pressure cooker at ~ 95–100 °C for 8 min for HMGA1 and 10 min for HAND1 with lids securely closed to maintain consistent pressure conditions. The sections were then cooled at room temperature (20–25 °C) and washed three times in 0.01 M PBS for 3 min each under gentle agitation. Primary antibodies against HMGA1 and HAND1 were diluted in antibody diluent (1:150 and 1:100, respectively) and mixed gently by pipetting before being applied evenly to completely cover each tissue section. The slides were incubated overnight at 4 °C in a humidified chamber. After overnight incubation, the slides were removed from the humidified chamber and allowed to equilibrate to room temperature for 10 min. Slides were washed three times with 0.01 M PBS (5 min per wash) under gentle agitation to remove unbound antibodies. Excess liquid was removed, and secondary antibody (HRP-labeled polymer anti-rabbit/mouse) was added to fully cover the tissue section surfaces. Further incubation was performed at 37 °C for 35 min.
DAB substrate solution was applied dropwise to cover the tissue, and color development was monitored under a microscope for 1–3 min until brown staining appeared and stopped immediately by rinsing with distilled water once optimal staining intensity was achieveds. Hematoxylin re-staining, hydrochloric acid alcohol differentiation, and ammonia solution for blue reversal were performed. This was followed by routine graded alcohol dehydration, xylene transparency, and mounting with neutral balsam for observing under a microscope. PBS was used as a blank control in place of the primary antibody. In addition, previously confirmed HMGA1-positive head and neck squamous carcinoma tissue sections and HAND1-positive placental tissue sections were used as positive controls to ensure staining quality and consistency.
Result determination
The chromogen used in this experiment is the DAB-H2O2 system. After hematoxylin re-staining, HMGA1 and HAND1 positive staining is mainly observed in the cell nucleus, with some cytoplasmic staining. For evaluation, the region with the highest density of positively stained cells was identified under low magnification (×40). Under ×100 magnification, five fields with the highest positive cell density were selected, and 100 cells were counted in each field. The average percentage of positive cells from the five fields is used as the result for each slide. The grading criteria were as follows: no stained cells (-), positive cells < 25% (+), positive cells 25%–50% (++), and positive cells > 50% (+++), based on a semi-quantitative percentage scoring method commonly used in immunohistochemical analyses. Evaluation was performed independently by two qualified pathologists in a double-blind manner15,16,17. If their opinions differ, they should discuss and reach a conclusion18.
RT-PCR method
Primers
Primers were designed using primer design software based on target gene sequences, with attention to primer specificity, melting temperature compatibility, and expected product size. The detailed primer sequences, product sizes, and annealing temperatures are summarized in Table 1. The primers were synthesized to a concentration of 2 OD, divided into two tubes, and diluted to 10 pmol/µL with DEPC-treated water for storage at -20 °C. The annealing temperature was calculated using the formula: Tm= 2(A + T) + 4(G + C)
RNase treatment of experimental materials and equipment:
Plastic Items (e.g., pipette tips, EP tubes): The plastic items were placed in a sterilized wide-mouth reagent bottle, prepared DEPC-treated water was added until the items were fully submerged, and the materials were incubated overnight in a fume hood at room temperature (20–25 °C). They were then sterilized under pressure for 120 min and dried at 65 °C.
Glass Items: The glassware was soaked overnight in acid, rinsed thoroughly, dried, wrapped in aluminium foil, and baked in a 200 °C oven for 6 h and stored in a clean place for future use.
Metal Items (e.g., scissors, tweezers): The metal items were cleaned, baked in a 200 °C oven for 6 h and stored in a clean place for future use.
Preparation of solutions
0.1% (v/v) DEPC Solution: 1 mL DEPC was added to 1000 mL distilled water to achieve a final concentration of 0.1% (v/v), shaken well, incubated at 4 °C overnight, sterilized by autoclaving, and stored at 4 °C.
75% (v/v) Ethanol Solution: 75 mL absolute ethanol was mixed with 25 mL DEPC-treated water to achieve a final concentration of 75% (v/v), and stored at 4 °C.
Total RNA extraction from tissue
Following RNA extraction, RNA integrity was assessed prior to reverse transcription and downstream analysis. 80 mg of tissue was weighed using an electronic balance, cut into small pieces, and placed in a pre-treated glass homogenizer. 1 mL pre-cooled RNA extraction reagent was added at 4 °C, and the sample was homogenized on ice using repeated grinding until a uniform lysate without visible tissue fragments was obtained, avoiding bubble formation. The homogenized mixture was transferred to a 1.5 mL RNase-free EP tube and incubated on ice for 5 min. 400 µL chloroform was added to each tube, vortexed vigorously for 10–15 s to ensure complete phase mixing, followed by incubation on ice for 5 min. The tubes were centrifuged at 4 °C, 12,000 x g for 15 min. After centrifugation, the upper aqueous phase was carefully aspirated using an RNase-free pipette without disturbing the interphase layer into a new tube. An equal volume of isopropanol was added, mixed by gently inverting the tube 8-10 times until the layers disappeared, and incubated at -20 °C for at least 10 min. The mixture was centrifuged at 4 °C, 12,000 x g for 10 min. The supernatant layer was discarded, 400 µL of 75% ice-cold ethanol was added to suspend the RNA pellet. The mixture was again centrifuged at 4 °C, 12,000 x g. for 5 min. The supernatant layer was discarded, and the RNA pellet was slightly dried. The RNA pellet was dissolved in RNase-free water and stored at -80 °C for future use.
RNA Integrity detection
For RNA integrity detection, 5 µL of RNA sample was mixed with 5 µL of bromophenol blue loading buffer, and electrophoresis was performed on a 2% agarose gel. RNA integrity was considered acceptable when clear 28S, 18S, and 5S bands were visible without obvious smearing.
Reverse transcription (RT) to synthesize cDNA
6 µL of RNA was preheated at 70 °C for 10 min, then 25 mM ＭgCl2 (4.0 µL), dNTP (2.0 µL), AMV (0.7 µL), 10× AMV Buffer (2.0 µL), oligo(dT) (1.0 µL), RNasin (0.5 µL) and deionized water were added sequentially on ice to achieve a final reaction volume of 20 µL, followed by gentle mixing and brief centrifugation. The reaction conditions were 25 °C for 1 min, 42 °C for 60 min, 98 °C for 5 min, and then stored at 4 °C indefinitely. Once the reaction reached 4 °C, the product was taken out of the PCR machine, used as a cDNA template for the next PCR reaction, and stored at -20 °C for later use.
PCR amplification
For each PCR reaction, the mixture contained cDNA template (1 µL), forward primer specific for the HMGA1, HAND1, or GAPDH (1 µL), reverse primer specific for the target gene or GAPDH (1 µL), PCR master mix (10 µL), and DEPC-treated water (7 µL), for a total volume of 20 µL. Separate reactions were prepared for each target gene (HMGA1, HAND1) and the internal control gene (GAPDH), with each reaction containing its respective gene-specific forward and reverse primer pair.
The total reaction volume was 20 µL. After gently mixing the reaction components and briefly centrifuging for 5–10 s at low speed, the tube was placed in a preheated PCR machine and amplified according to the reaction conditions in Table 2.
Confirmation and analysis of PCR amplified products
Gel preparation
2 g of agarose was weighed and placed in a triangular flask. 2 mL of 50× TAE electrophoresis buffer and 100 mL of distilled water (prepare a 2% agarose gel) were added, then heated in a microwave oven with intermittent swirling (15–20 s intervals) until the agarose was completely dissolved. After cooling to approximately 55 °C, 10 µL of EB solution was added and mixed thoroughly. The gel was then poured into a clean gel tray at room temperature (20–25 °C), washed with distilled water, and a comb was inserted to create sample wells. The gel thickness was 0.4 cm. The tray was placed on a flat surface at room temperature (20–25 °C) for 30 min until the gel solidified completely. The comb was carefully removed.
Loading samples
4 µL of each DNA sample was added into the wells of the gel, and DNA Marker was added to the leftmost well.
Electrophoresis
The loaded gel tray was placed into an electrophoresis tank filled with TAE electrophoresis buffer, ensuring that the buffer level remained about 1 mm above the gel surface. One end of the wells was at the cathode (to allow DNA to migrate toward the anode). The electrophoresis system was connected to a power supply and run at 120 V and 60 mA for approximately 45 min, until the loading buffer migrated beyond the midline of the gel, after which the power was turned off.
Result observation and imaging
After electrophoresis, the gel was carefully removed, and the results were observed under a UV transilluminator and compared with the DNA Marker to determine whether the molecular weight of the amplified fragments appeared at the expected positions. The gel imaging system was used to capture images and save the results on the computer.
Control setup
Distilled water was used instead of cDNA as a negative control.
Semi-quantitative analysis
The agarose gel electrophoresis results were analyzed and captured using the gel imaging system. Image analysis software was used to analyze the brightness of the bands. The absolute integrated OD values of the target gene amplification band and the corresponding internal control gene amplification band were measured. The results were expressed as the ratio of the absolute integrated OD value of the target band to the absolute integrated OD value of the corresponding internal control band.
That is, relative expression = target gene OD value / internal control gene OD value.
Statistical analysis
All data were organized using Excel and analyzed using statistical software. Continuous variables were expressed as mean ± standard deviation (SD) and compared between two groups using the independent-samples t-test. Nominal categorical variables, including immunohistochemical positive expression rates, were analyzed using the χ2 test. Ordinal categorical variables, including pathological grade and clinical stage, were analyzed using the rank-sum test (Mann-Whitney U test for two-group comparisons, where applicable). Correlation between HMGA1 and HAND1 expression was evaluated using linear correlation analysis. All tests were two-tailed; P < 0.05 was considered statistically significant, and P. < 0.01 was considered highly significant.
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Immunohistochemical detection results
Representative hematoxylin–eosin (HE) staining images illustrating the histological morphology of adjacent normal laryngeal mucosa and LSCC with varying degrees of differentiation (well, moderate, and poor) are shown. Specifically, Figure 1A shows adjacent normal laryngeal mucosa, Figure 1B shows well-differentiated LSCC, Figure 1C shows moderately di...
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Laryngeal cancer, one of the most common tumors of the head and neck, has shown an increasing incidence in recent years, likely associated with environmental and lifestyle-related factors. Like other malignant tumors, its recurrence and metastasis remain the leading causes of death. With advancements in molecular biology, many factors associated with the development of laryngeal cancer have been identified, but the mechanisms of onset and metastasis are still not fully understood. HMGA1, as a transcriptional regulator, h...
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The authors would like to acknowledge the support of their institution and all individuals who contributed to this study.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Automatic Tissue Embedding Machine | Sakura Finetek, Tokyo, Japan | Tissue-Tek TEC 5 | Paraffin embedding |
| Microtome | Leica Microsystems, Wetzlar, Germany | RM2135 | Sectioning (4 μm) |
| Slide Dryer | Xiaogan Hongye Medical Instrument Co., Ltd., China | CS-VI | Slide drying |
| Heating Incubator | Shanghai Yangguang Experimental Instrument Co., Ltd., China | 303-4A | Temperature-controlled incubation |
| Optical Microscope | Olympus Corporation, Tokyo, Japan | BX41 | Histological observation |
| Image Capture System | QImaging, Canada | QCapture Pro 7 | Microscopy imaging |
| Clean Bench | Beijing Xicheng Semiconductor Equipment Factory, China | SW-CJ-1FD | Sterile workspace |
| Refrigerated Centrifuge | Beijing Medical Instrument Repair Factory, China | LXJ-64-01 | RNA separation |
| Tabletop Centrifuge | Shanghai Peiqing Technology Co., Ltd., China | PQ-1600A | RNA/DNA processing |
| Magnetic Stirrer | Jiangsu Jintan Medical Instrument Factory, China | HJ-3 | Solution preparation |
| Mini Mixer | Shanghai Peng’s Industrial Co., Ltd., China | H-1 | Sample mixing |
| Electronic Balance | METTLER Toledo, Switzerland | PB3002-S | Sample weighing |
| PCR Amplifier | Eppendorf AG, Germany | Mastercycler 5333 | PCR amplification |
| Electrophoresis Power Supply | Beijing Liuyi Instrument Factory, China | DYY-6C | Voltage control |
| Electrophoresis Tank | Beijing Liuyi Instrument Factory, China | DYCZ-24DN | Gel electrophoresis |
| Microwave Oven | Shanghai Zhongda Medical Application Research Institute, China | WD800SL23 | Agarose preparation |
| UV Transilluminator | Beijing Liuyi Instrument Factory, China | WD-9403B | DNA visualization |
| Gel Imaging System | Anlai Co., Ltd., China | AlphaImager HP | Gel documentation |
| Refrigerator (4 °C) | Haier Biomedical, China | BC-165D | Sample storage |
| Ultra-Low Temperature Freezer | Haier Biomedical, China | DW-86L388 | Storage (-80 °C) |
| Ice Box | Shanghai Fuzhi Technology Co., Ltd., China | FZ-IB-20L | Sample transport |
| Cryogenic Storage Bags | Shanghai Yili Technology Co., Ltd., China | YL-ULT-50 | Sample preservation |
| Research Pipettes | Eppendorf AG, Germany | Research Plus | Liquid handling |
| RNA Extraction Reagent (TRIzol) | Invitrogen (Thermo Fisher Scientific), USA | 15596026 | RNA extraction |
| Reverse Transcription Kit | Takara Bio Inc., Japan | RR037A | cDNA synthesis |
| PCR Master Mix | Takara Bio Inc., Japan | RR820A | PCR amplification |
| dNTP Mix | Takara Bio Inc., Japan | 4030 (typical) | PCR component |
| RNase Inhibitor (RNasin) | Promega, USA | N2111 | RNA protection |
| Oligo(dT) Primer | Takara Bio Inc., Japan | 3806 (typical) | cDNA synthesis primer |
| Agarose | Sigma-Aldrich, USA | A9539 | Gel preparation |
| TAE Buffer (50×) | Sigma-Aldrich, USA | TAE50X-1L (typical) | Electrophoresis buffer |
| DNA Marker (Ladder) | Takara Bio Inc., Japan | DL2000 (3421A) | Size reference |
| Ethidium bromide (EtBr) | Bio-Rad, USA | 1610433 | DNA visualization |
| PBS Buffer | Gibco (Thermo Fisher Scientific), USA | 10010023 | Washing buffer |
| DEPC-treated Water | Invitrogen, USA | AM9916 | RNA protection |
| Chloroform | Sigma-Aldrich, USA | C2432 | RNA extraction |
| Isopropanol | Merck, Germany | 109634 | RNA precipitation |
| Ethanol (absolute) | Merck, Germany | 100983 | Dehydration |
| Methanol | Merck, Germany | 106009 | Peroxidase blocking |
| Hydrogen Peroxide (H2O2) | Sigma-Aldrich, USA | H1009 | Endogenous peroxidase blocking |
| Xylene | Sigma-Aldrich, USA | 534056 | Deparaffinization |
| Citrate Antigen Retrieval Buffer | Abcam, UK | ab93678 | Antigen retrieval |
| Antibody Diluent | Dako, Denmark | S3022 | Antibody dilution |
| HMGA1 primary antibody | Abcam, UK | ab129153 | Dilution 1:150 |
| HAND1 primary antibody | Abcam, UK | ab200040 | Dilution 1:100 |
| Secondary antibody (HRP-conjugated) | Dako, Denmark | K5007 | Detection system |
| DAB Substrate Kit | Dako, Denmark | K3468 | Chromogenic detection |
| Hematoxylin Stain | Sigma-Aldrich, USA | H9627 | Nuclear staining |
| Eosin Stain | Sigma-Aldrich, USA | E4009 | Cytoplasmic staining |
| Neutral Balsam Mounting Medium | Sinopharm Chemical Reagent Co., China | 10004160 | Slide mounting |
| HMGA1 Primers | Custom synthesized (e.g., IDT/Sangon) | N/A | Gene amplification |
| HAND1 Primers | Custom synthesized (e.g., IDT/Sangon) | N/A | Gene amplification |
| GAPDH Primers (internal control) | Custom synthesized | N/A | Housekeeping gene |
| APES (Aminopropyltriethoxysilane) | Sigma-Aldrich | N/A | Slide coating |
| RNase-free Microcentrifuge Tubes | Eppendorf | N/A | RNA handling |
| RNase-free Pipette Tips | Eppendorf | N/A | Liquid handling |
| Agarose Gel Casting Tray and Comb | Generic | N/A | Gel preparation |
| Primer Design Software | Integrated DNA Technologies, USA | PrimerQuest Tool | Primer design |
| Image Analysis Software | Media Cybernetics, USA | Image-Pro Plus 6.0 | Band quantification |
| Statistical Analysis Software | IBM Corp., USA | SPSS Version 13.0 | Statistical analysis |